Leader: Federico Carpi (UNIFI); Other collaborator(s):
This task has the following goals: (i) Development of extracorporeal perfusion systems for donor organs, so as to enable ex-vivo investigations on aging at an organ level; this will allow for testing directly on perfused organs the response to drugs normally administered to the elders (e.g., anti-inflammatory, metabolic or immunosuppressive molecules). (ii) Development of cardiac organoids, for in-vitro investigations on aging at a tissue level; this activity has the long-term vision of testing personalised drugs on 3D cellular constructs based on cardiomyocytes that are derived from human induced pluripotent stem cells (iPSC), which can be obtained by harvesting primary cells directly from the patient that requires the personalised drug.
During the concerned period, the research activities have covered the following aspects:
- The activities on the development of an extracorporeal perfusion device for donor organs have benefited from the entry into service of a new Researcher, who designed and developed an electronic circuit to control and monitor the energy absorption of the heat exchanger during the perfusion process.
- The activities on the development of cardiac organoids have been focused on the development of a biocompatible hydrogel that can host human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, suitable for 3D bioprinting.
Main policy, industrial and scientific implications:
The research activities concerned by this Task could have the following important implications:
- testing on perfused ex-vivo organs the response to drugs normally administered to the elders;
- in-vitro testing of personalised drugs on 3D cellular constructs based on cardiomyocytes having the same genes of the patient, as they can be derived from human induced pluripotent (iPSC) stem cells, which can be obtained by harvesting primary cells directly from the patient.
Please see the next reporting period.
During the concerned period, the research activities have covered the following topics:
1) Development of an extracorporeal perfusion device for donor organs: The activities were focused on the design, production, and testing of a new heat exchanger based on Peltier cell technology. The device allowed for smoothly changing the temperature of the perfused organ from 4°C to 37°C, and to reverse the process from 37°C to 4°C, without the use of ice. Tests on ex-vivo animal organs were conducted during the normothermic perfusion phase using the device. The results (which are accepted for publication in the IEEE Open Journal of Engineering in Medicine and Biology), demonstrated the device's ability to rewarm and maintain the organs at a nearly constant temperature (33°C) for an average of 4 hours, without causing the severe damage typically observed in organs stored in ice without perfusion. In these experiments, a 2-hour hypothermic perfusion (4°C) was performed using a commercial machine in combination with ice. The goal of future experiments is to use the developed device for both the phases of the protocol (hypothermic and normothermic), to reduce the number of machines used in the transplant procedure and optimize temperature control, thereby minimizing temperature fluctuations and extending perfusion time to up to 12 hours.
2) Development of cardiac organoids: The activities were focused on the creation of a biocompatible hydrogel that can host human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, suitable for 3D bioprinting. In particular, a 3D-printable gelatin methacrylate-xanthan gum hydrogel bioink was developed and characterised. Tests with hiPSCs showed the hydrogel’s ability to promote their proliferation within both 2D and 3D cell cultures. The tests also showed that hiPSCs inside hemispheres of the hydrogel were able to differentiate into cardiomyocytes, capable of spontaneous contractions (average frequency of ~0.5 Hz and amplitude of ~2%). Furthermore, bioprinting tests proved the possibility of fabricating 3D constructs of the hiPSC-laden hydrogel, with well-defined line widths (~800 mm). The work was presented at the IDBN 2024 National Congress (26-27 September 2024, Florence, Italy). A research paper on this work was published in the Journal of Functional Biomaterials.
During the concerned period, the research activities covered the following topics:
- Development of an extracorporeal perfusion device for donor organs: The activities focused on defining the details of the new experimental clinical protocol (up to 12h), with the aim of assessing the viability of perfused organs using an innovative dataset. This dataset combines immune histological analysis of tissue samples with histochemical analysis of the perfusion liquid. Official authorization was obtained from USL Toscana Centro to collaborate with slaughterhouses to optimize the organ explant protocol. This collaboration aims to minimize damage to the organs and preserve vessel integrity during the procedure. Additionally, a storage method for transportation by car to the Simulation Lab (Careggi AOUC, Florence) was established to ensure the preservation of organ integrity. New certified disposable pressure sensors for clinical use were selected and will be integrated into the firmware in the next phase of development. The enclosure for the Control PCB was redesigned to ensure efficient ventilation for all PCB components and to stabilize the operating temperature during long perfusion cycles (up to 12 hours). A paper detailing the results of previous experiments was published: “Experimental Evaluation of a New Perfusion Machine using Normothermic Cycles on Explanted Livers” (https://dx.doi.org/10.1109/OJEMB.2024.3478791). Furthermore, the pre-incubation phase of the FLOWeR Project, conducted at the Florentine Incubator, has concluded. This project aims to develop a UNIFI spin-off based on the innovative perfusion machine. In the coming months, potential investors will be engaged. The goal of future experiments is to use the developed device in both phases of the protocol (hypothermic and normothermic). Research is ongoing to select a commercial (FDA approved or under examination) perfusion solution capable of substituting blood in carrying oxygen to cells during prolonged normothermic phases.
- Development of cardiac organoids: The activities on this topic during the last period were focused on deepening the investigation on the properties of a biocompatible hydrogel developed by our group to host and 3D print human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. The hydrogel consists of a gelatin methacrylate-xanthan gum composite. While initial tests on 2D and 3D cultures of hiPSCs in the hydrogel had shown the material’s ability to promote hiPSC proliferation and differentiation into cardiomyocytes, extensive trials of 3D printing the bioink during this period have not produced the desired effects, so far. In particular, the cells have been found not to be viable after the 3D printing process. We are currently investigating possible reasons, by systematically varying printing parameters and conditions, as well as by modifying the hydrogel composition.
During the concerned period, the research activities covered the following topics:
- Development of an extracorporeal perfusion device for donor organs: Experimental sessions were conducted using the developed perfusion device, confirming the feasibility of the developed retrieval and perfusion protocol. Animal livers, collected from the slaughterhouse in Chiesina Uzzanese (PT) under the supervision of ASL veterinarians, were washed with a physiological solution according to the protocol; after appropriate storage in ice within a thermal container, they were transported to the Simulation LAB at the Surgical Clinics of Careggi Hospital. Following the surgical procedure of cannulating the arterial and venous vessels, the livers were placed in the perfusion machine and subjected to different thermal protocols to evaluate the stability of physiological and chemical parameters. The machine was tested in all its functions (centrifugal pumping, peristaltic pumping, filtering, oxygenation, thermal maintenance, energy consumption). During the experiments, chemical tests were conducted using blood gas analysis (Emogas) to track the time-course of parameters such as dissolved oxygen in the perfusion fluid, lactate levels released by the organ as an indicator of general inflammation, pH, and other specific values. Thanks to endurance experiments lasting over 12 hours, conducted with both hypothermic and normothermic cycles, it was possible to test both the selected components and the logic of the perfusion machine's circulation system. The machine proved to be capable of maintaining thermal, pressure, oxygenation, and filtration parameters within ranges suitable for the survival of a perfused porcine liver. For these initial experiments, a physiological solution was used as the perfusion fluid, yielding better preservation results with hypothermic cycles. For each experiment, a histological sampling campaign was conducted, which in the coming months will allow for the analysis of the quality of the perfused organs using fluorescence immunohistology examinations under different customized thermal protocols.
- Development of cardiac organoids: The activities on this topic during the last period had to cope with a severe limitation, due to the breakdown of the 3D bioprinter that had been used so far for this research. As a result, 3D printing was (and still is) prevented. While waiting for the possibility of using the instrumentation again, the research has been focused on investigating commercial biocompatible UV-curable hydrogels (such as LunaGel by Gelomics), as possible suitable carriers for human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Such cells were found to be viable after mixing with LunaGel, and undergoing droplet deposition and UV curing, so as to obtain 3D (hemispherical-like) structures. This provided evidence that LunaGel can effectively be used with hiPSC-derived cardiomyocytes to create 3D gel structures hosting vital cardiomyocytes.
During the concerned period, the research activities covered the following topics:
- Development of an extracorporeal perfusion device for donor organs: More experimental sessions were conducted using the developed perfusion device, to increase the number of samples collected for the histological analysis and to test the endurance of the device. Following the surgical procedure of cannulating the arterial and venous vessels, the livers were placed in the perfusion machine and subjected to different thermal protocols to evaluate the stability of physiological and chemical parameters. For these experiments, a Belzer UW MPS® machine perfusion solution was used as the perfusion fluid, to highlight the comparison with the previous experiments and better preservation results with hypothermic cycles. The viscosity of the solution requested a new calibration of the sensors and pumps to obtain the best perfusion. For each experiment, a histological sampling campaign was conducted, which in the coming months will allow for the analysis of the quality of the perfused organs using fluorescence immunohistology examinations under different customized thermal protocols. The following histological analyses were performed on the collected samples: Haematoxylin and Eosin, PAS, Picrosirius Red, and immunofluorescence with CD31. In the next period, additional immunofluorescence analysis will be performed to estimate the micro and macro scale of tissues damage after the different thermal protocols. The design of the machine was presented to the patent commission of University of Florence on the 3rd of June to protect the technical solutions developed by the inventors. The commission approved on the 1st of July the procedure to instruct the deposit of a Patent application.
- Development of cardiac organoids: The activities on this topic during the last period were focused on the design and preliminary experiments to create a heart chamber organoid with human iPSC cells. The idea is to create a deformable chamber around a plastic balloon, to be coated with a hydrogel, loaded with iPSC cells to be differentiated into cardiomyocytes. While the chamber’s deformation will be ensured by an external fluidic system (which will pump a fluid into and out of the balloon), the goal is to investigate how the iPSC cells will be able to differentiate into cardiomyocytes and organise a tissue around the dynamically expanding balloon, which will therefore act as a dynamic permanent scaffold. To enable an implementation of this concept, a custom bioreactor is being designed and fabricated on purpose.
During the concerned period, the research activities covered the following topics:
- Development of an extracorporeal perfusion device for donor organs: The development of a new extracorporeal perfusion device (BMP) for donor organs required a thorough evaluation of tissue quality under different preservation protocols, compared against traditional Static Cold Storage (SCS). Histological samples were collected, processed, and analyzed using a dedicated software tool developed in Python with OpenCV, capable of extracting quantitative data on tissue conservation. The study focused on livers subjected to three experimental conditions: a 10-hour D-HOPE perfusion with physiologic solution, a 10-hour D-HOPE perfusion with Belzer UW MPS solution, and a 10-hour D-NOPE perfusion also with Belzer UW MPS solution. After collection, each tissue sample was stored in 30% sucrose in PBS, embedded in OCT Killik, and preserved at –80 °C. From each sample, ten slides were prepared and stained with hematoxylin and eosin; ten images were acquired per slide and subsequently analyzed quantitatively through the custom software. The analysis allowed for monitoring the temporal evolution of three key histological components: acidophilic structures, basophilic structures, and other tissue elements. Preliminary results clearly demonstrate that the Biodynamic Perfusion Machine (BPM) outperforms static cold storage, providing better overall tissue preservation. Furthermore, the comparison between solutions highlighted that perfusion with physiologic solution is less effective at maintaining tissue homogeneity than perfusion with Belzer UW MPS, which showed superior stability. These encouraging findings pave the way for further investigations. In the coming months, additional experiments will employ immunofluorescence techniques to deepen correlations between perfusion protocols and quantitative histological outcomes, with the goal of definingstronger and more objective criteria for organ quality assessment.
- Development of cardiac organoids: In the last period, we developed the first prototype of a deformable hydrogel-based cell scaffold to be used to create a cardiac chamber organoid with human iPSC cells. The construct was obtained using an agarose hollow mold and an inflatable small medical-grade silicone balloon, on top of which the gel was cross-linked. The balloon was later removed, obtaining a hollow hydrogel chamber. Different hydrogel formulations were tested, until a suitable tradeoff (5% fibrinogen, 3% thrombin, 5% Matrigel) was found to ensure a stable and uniform scaffold. A water-based pressurization test showed the chamber’s ability to sustain deformation without breaking. During the next period, the chamber will be further developed, targeting an integration with iPSC cells and evaluating cell viability and proliferation within the matrix. Comparative studies will determine whether encapsulating pre-differentiated cardiomyocytes or undifferentiated iPSCs is more effective.
During the concerned period, the research activities covered the following topics:
- Development of an extracorporeal perfusion device for donor organs: Additional histological analyses were conducted using Periodic Acid-Schiff (PAS) staining to detect glycoproteins and glycogen. The effect of machine perfusion (MPS) diverged markedly in the presence of polysaccharides from static cold storage (SCS) by the end of the comparison cycles (MPS ~76%; SCS ~67%). This finding reflects sustained metabolic activity rather than cellular quiescence and is consistent with reduced cellular stress and lower activation of injury pathways. Further histological evaluation using Picrosirius Red staining was performed to assess fibrosis and collagen deposition in the tissue. At the short-term time point (12 h), collagen levels showed minimal variation between MPS (~87%) and SCS (~84%), suggesting an adaptive or early fibrogenic response under both conditions rather than established fibrosis. This may have implications for tissue stiffness and microcirculatory function. Additional immunohistochemical analyses were conducted with a primary anti-CD31 antibody and a FITC-conjugated anti-rabbit secondary antibody, in collaboration with the Department of Biology. These analyses provided relevant insights regarding vascular integrity: endothelial cell density remained largely unchanged between SCS and MPS over 12 hours, with overlapping variability across time points. This indicates preserved endothelial structural integrity. All images were quantitatively analysed using ImageJ software and validated by Prof. Stefano Bacci, a histological expert involved in the research. In conclusion, the proposed methodology, which integrates the developed Experimental Protocol with the novel extracorporeal perfusion device (Biodynamic Machine Perfusion -BMP) and leverages dedicated software for histological analysis, allows for a quantitative evaluation of tissue preservation quality and supports future comparisons across different preservation cycles. The results confirm the superior effectiveness of MPS over SCS when applied using this protocol.
- Development of cardiac organoids: In the last period, cell viability and proliferation were assessed within the previously selected gel formulation (5% fibrinogen, 3% thrombin, 5% Matrigel) for application in a deformable hydrogel-based scaffold aimed at generating a cardiac chamber organoid. hiPSCs were seeded at different densities in gel droplets; in all conditions tested, they exhibited robust proliferative capacity, organizing into spheroidal colonies. Differentiation of hiPSCs within the gel into cardiomyocytes was possible, as evidenced by spontaneous contractions, but was reproducible only at low cell densities. In parallel, preliminary mechanical characterizations of the gel were performed, and a first prototype of the deformable scaffold containing gel and hiPSCs was developed, showing an overall homogeneous cell distribution. However, crosslinking times need to be reduced to better preserve cell viability. During the next period, further experiments with hiPSCs will be conducted to optimize the protocol, followed by the introduction of pre-differentiated cardiomyocytes.
- Deidda, V., Ventisette, I., Langione, M., Giammarino, L., Pioner, J.M., Credi,C., Carpi, F. (2024). 3D-printable gelatin methacrylate-xanthan gum hydrogel bioink enabling human induced pluripotent stem cell differentiation into cardiomyocytes. Journal of Functional Biomaterials, 15(10), 297. DOI: 10.3390/jfb15100297.
- E. Barcali, L. Maggi, R. Panconesi, F. Staderini, L. Bocchi, C. Nardi, N. Navari, A. Peris, M. Risaliti, M. F. Carvalho, F. Marra, P. Dutkowski, G. L. Grazo, A. Schlegel, F. Bigi, and M. Dimitri, A. Corvi. Experimental Evaluation of a New Perfusion Machine using Normothermic Cycles on Explanted Livers. IEEE Open Journal of Engineering in Medicine and Biology. In press.
- Mattia Dimitri, “F.L.O.WeR. (For Life Organ We Research): A Next-Generation of Biodynamic Machine Perfusion (BMP) for organ preservation to promote healthy longevity”, Age-It General Meeting, 29-31 Ottobre 2025, Napoli.