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Supercritical carbon dioxide for delipidation of soft tissue - Suppressing transplant rejection.

Periodic Reporting for period 1 - CriTiClean (Supercritical carbon dioxide for delipidation of soft tissue - Suppressing transplant rejection.)

Reporting period: 2017-05-22 to 2019-05-21

The lack of donor organs and tissue constitutes a health and social problem. Even though the global rate of transplantation increased by 7.25% between 2015 and 2016, less than 10% of the transplant needs are covered, which results in a high unbalanced supply-and-demand rate. A failed transplantation process is mainly due to the presence of cellular components in the donor tissue.
Biological extracellular matrix (ECM) technology, aims to obtain functional scaffolds from animal sources, where cellular components are removed while keeping the mechanical and architectonic characteristics of the ECM. Mentioned cellular components to be removed are lipids and genetic material. Lipids residues may hamper ECM production as certain lipids impairs recellularization by poor cell attachment and they may act as a source of pro-inflammatory fatty acids. Moreover, residual antigens (dsDNA) should be minimized to avoid the activation of the host immune system.
Traditional decellularization methods are based on surfactant-enzyme treatments. Such processes are high time consuming and were proven to damage the ECM by fibre’s break and/or crosslinking. Moreover, detergent leftovers may present toxic effects in the host. Therefore, there is a need of new alternatives to produce suitable ECM scaffolds which avoid the use of detergents while ensuring a safer transplantation process.
CriTiClean was conceived to offer new alternatives to the worldwide transplantation issue. This project focused in the exploration of pressurize CO2-based fluids to remove lipids and, if possible, other cellular components from donor tissues. CriTiClean approach avoids the use of chemicals, avoiding the side effects of traditional processes. This newcoined biomedical application of pressurized CO2-based technology gained the attention of the scientific community in the last 10 years. Therefore, very scarce information related to the effect of such technology on the tissue lipid composition after treatment can be found in the literature. CriTiClean action plan was based on an exhaustive delipidation study from an analytical chemistry point of view, while keeping the interest in dsDNA removal and preservation of ECM integrity.
At the term of CriTiClean, the hypothesis set in the action was confirmed, with few restrictions: certain pressurized CO2-based fluids are able to remove lipids and other cellular components (after enzyme treatment) from porcine soft tissues, without jeopardizing the structure and architecture characteristics of the native tissue. However, the presence of immune reactive components (dsDNA) is still an ongoing issue. In conclusion, CriTiClean outcomes proved that this new decellularization approach deserves to be continued in future projects.
Action´s team members planned and designed a specific device (insert) for the pressurized CO2-based treatment of biological soft tissues, able to resist high pressures and temperatures maximizing the diffusion of the fluid into the tissue while ensuring its integrity (Figure 2). An effective lipid extraction and analysis methodology was achieved using pulmonary arteries as a tissue model. Most used methods for lipid extraction based on solid-liquid methodologies, were tested. Ultrahigh performance supercritical chromatography (UHPSFC) coupled to quadrupole time of flight (MS/MS) were used for an accurate identification/quantification of lipids from the biological material.
Porcine tissues, pulmonary arteries, retina and cava veins, were submitted to neat supercritical CO2 (scCO2) and other pressurized CO2-based mixtures, i.e. using ethanol-water and limonene. Design of Experiments (DoE) tool was used to cover a wide range of pressurized conditions, considering temperature, time and pressure as factors. Lipids remaining in the tissues were identified and quantified by the previously developed analytical methodology. Clustering studies to elucidate the connections between extraction conditions fluid and lipid classes/species and dsDNA removal were also performed. Statistic tools such as common factor analysis and principal component analysis (PCA) were used for a exhaustive study and visualization of the obtained results. Alternative approaches for a successful dsDNA removal were addressed, i.e. pressurized hot water extraction or electroporation. Histological analysis of treated samples was performed to determine the dsDNA presence (by nuclei detection), the overall extracellular matrix structure and the integrity of elastic fibres.

Pressurized CO2-based mixtures constitute a powerful tool for biological tissues decellularization, i.e. pulmonary arteries, cava veins and retina from pigs. The composition of the pressurized fluids was proven to be crucial for a satisfactory delipidation, affecting to the efficiency and selectivity of the process. Pressurized CO2-limonene mixtures showed to enhance a further enzymatic activity. The ECM analysis of treated samples showed that its structure and architecture was preserved, resulting similar to those from native tissues and even better when detergents-enzymes combination were used.
At the beginning of the action, a couple of publications claimed the effectivity of this technology in biological tissues decellularization. From the starting date of CriTiClean (2017), new research groups around the world (eight so far) have shown their achievements in the field. CriTiClean increased the knowledge of this novel biomedical application of pressurized CO2-based fluids.
In particular, this action contributed to:
- the lipid composition of several biological tissues from pigs, i.e. pulmonary arteries, retina and cava veins.
- the selectivity and efficiency of lipid removal by pressurized CO2-mixtures, especially when ethanol-water or limonene were added.
- the poor efficiency of neat supercritical CO2 to remove lipids. Only triacylglycerides content was decreased.
- the correlation between delipidation and the fluid composition.
- the high delipidation efficiency of pressurized CO2-ethanol-water .
- the positive dsDNA removal effect of pressurized CO2-limonene mixtures by a tentative enhancement of the further enzymatic activity (dsDNAses).
- the unsuccessful results related to dsDNA removal from electroporation or pressurized hot water extraction (with/without benzoases) before and after pressurized CO2-mixtures approaches.
- the new suitable alternative based on sequential CO2-based mixtures extractions for both lipids and dsDNA removal in one step (data are currently being analyzed).
- the preservation of ECM integrity after pressurized CO2-based extractions.

CriTiClean dissemination allowed us to set new collaborations, which shows the increasing interest of the scientific community. On May 2019, Dr. Gil-Ramírez was interviewed by Mrs. Aisling Irwin, a science journalist of ´Horizon, the EU Research and Innovation magazine´, which resulted in the publication ´Transplant recipients may soon have a test to protect against organ rejection´(https://horizon-magazine.eu/article/transplant-recipients-may-soon-have-test-protect-against-organ-rejection.html). CriTiClean is expected to be a prequel of new projects.