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.
- 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.