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. 2021 Dec 15:9:796157.
doi: 10.3389/fbioe.2021.796157. eCollection 2021.

Development of a Novel Perfusable Solution for ex vivo Preservation: Towards Photosynthetic Oxygenation for Organ Transplantation

Valentina Veloso-Giménez  1 , Rosalba Escamilla  1   2 , David Necuñir  1 , Rocío Corrales-Orovio  1   3 , Sergio Riveros  4 , Carlo Marino  4 , Carolina Ehrenfeld  1   2 , Christian Dani Guzmán  5 , Mauricio P Boric  2 , Rolando Rebolledo  1   6 , José Tomás Egaña  1
Affiliations

Development of a Novel Perfusable Solution for ex vivo Preservation: Towards Photosynthetic Oxygenation for Organ Transplantation

Valentina Veloso-Giménez et al. Front Bioeng Biotechnol. .

Abstract

Oxygen is the key molecule for aerobic metabolism, but no animal cells can produce it, creating an extreme dependency on external supply. In contrast, microalgae are photosynthetic microorganisms, therefore, they are able to produce oxygen as plant cells do. As hypoxia is one of the main issues in organ transplantation, especially during preservation, the main goal of this work was to develop the first generation of perfusable photosynthetic solutions, exploring its feasibility for ex vivo organ preservation. Here, the microalgae Chlamydomonas reinhardtii was incorporated in a standard preservation solution, and key aspects such as alterations in cell size, oxygen production and survival were studied. Osmolarity and rheological features of the photosynthetic solution were comparable to human blood. In terms of functionality, the photosynthetic solution proved to be not harmful and to provide sufficient oxygen to support the metabolic requirement of zebrafish larvae and rat kidney slices. Thereafter, isolated porcine kidneys were perfused, and microalgae reached all renal vasculature, without inducing damage. After perfusion and flushing, no signs of tissue damage were detected, and recovered microalgae survived the process. Altogether, this work proposes the use of photosynthetic microorganisms as vascular oxygen factories to generate and deliver oxygen in isolated organs, representing a novel and promising strategy for organ preservation.

Keywords: Chlamydomonas reinhardtii; hypoxia; ischemia; organ perfusion; organ preservation; photosynthesis; photosynthetic microorganisms.

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Conflict of interest statement

Author CG was employed by company Sky-Walker SpA. Competing Interests: JTE is CSO and co-founder of SymbiOx Inc., a start-up company that owns IP in the field of this work. Thanks to an R and D grant provided by the Chilean Ministry of Economics (CORFO), during the conduct of this project, DN, RE and RC-O were full-time employees of SymbiOx Inc. All other authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Viability of C. reinhardtii in RLM. C. reinhardtii were incubated for 24 h in their culture media (TAP), a standard solution for organ preservation (RLM) or a mix of both in a 1:1 ratio (TAP:RLM). Viability of microalgae was not affected as shown by their growing capacity (A) and by flow cytometry (B). Data are expressed as mean ± SD; N = 3; ns: non-significant (one-way ANOVA test).
FIGURE 2
FIGURE 2
Morphology of C. reinhardtii in RLM. C. reinhardtii were incubated for 24 h in their culture media (TAP), a standard solution for organ preservation (RLM) or a mix of both in a 1:1 ratio (TAP:RLM). Morphology (A) and size (B) of the microalgae were not affected by the media. Arrow heads indicate size marker beads of 4, 6, 10, and 15 µm in diameter, from left to right. Scale bars represent 25 µm. Data are expressed as mean ± SD; N = 3; ns: non-significant (one-way ANOVA test).
FIGURE 3
FIGURE 3
Characterization of the photosynthetic solution. Different densities of C. reinhardtii were added to a standard solution for organ preservation (RLM). The oxygen production rate of microalgae was maintained after 24 h, except for 106 C. reinhardtii/ml, which increased (A). Up to 108 C. reinhardtii/ml, no significant differences were observed in the osmolality (B) and rheological properties of the solution (C). Zebrafish larvae were exposed for 24 h to photosynthetic solution containing different densities of microalgae, in the dark. Up to 108 C. reinhardtii/ml larvae presented normal phenotypes compared to control [E3; (D)]. Mild and severe mortality were observed at 108 and 109 C. reinhardtii/ml, respectively (E). Scale bars represent 1 mm in D. Data are expressed as mean ± SD; N = 3, 4; *p < 0.05, ***p ≤ 0.001 (one-way ANOVA followed by Tukey’s test in A; one-way ANOVA followed by Dunnett’s test in B; two-way ANOVA followed by Sidak´s test in C); different letters in E indicate significant differences with p < 0.05 (one-way ANOVA followed by Tukey´s test).
FIGURE 4
FIGURE 4
Oxygenation capacity of the photosynthetic solution. Oxygen concentrations were measured for 5 min in darkness (I, OFF) or light (II, ON). Then, microalgae were incorporated and measurements were performed for 10 min in the presence (III, ON) or absence (IV, OFF) of light (A). In the absence of light or microalgae (I, II and IV) a negative slope of the curve was seen for both, zebrafish larvae (B,C) and kidney slices (D,E), while in the presence of light and microalgae, the slope became positive for the larvae and nearly flat for the slices. A representative curve is shown for each experiment (B–D) and their metabolic rates calculated from the slopes (C–E). Data are expressed as mean ± SD; N = 3; different letters in C and E indicate significant differences with p < 0.05 (one-way ANOVA followed by Tukey´s test).
FIGURE 5
FIGURE 5
Distribution of the photosynthetic solution in porcine kidney. Compared to controls, organs turned green after perfusion [(A), left and right]. Fresh slices show a vascular distribution of the solution in the renal cortex [(B), top] and medulla [(B), bottom]. Cryosections of perfused kidneys show the distribution of C. reinhardtii in glomeruli and afferent arteriole [(C), top] and medullar blood vessels and capillaries [(C), bottom]. Scale bar represents 2 cm in A, 5 mm in (B) (top, left), 1 mm in (B) (top, right) and (B) (bottom, left), 250 µm in (B) (bottom, right), 100 µm in (C).
FIGURE 6
FIGURE 6
Dynamic perfusion of isolated porcine kidneys. Schematic representation of the ex vivo perfusion system, containing a pressure-flow controlling device, a centrifuge pump, and a container for the isolated organs (A). Vascular parameters were measured during the photosynthetic perfusion and the subsequent flushing step. Mean arterial pressure (MAP) was set to 70–80 mmHg remaining stable during the entire procedure (B). Perfusion flow decreased during the PSOP perfusion (C), while renal vascular resistance (RVR) increased, recovering during the flushing step (D). Data are expressed as mean ± SEM; N = 3 in (B–D).
FIGURE 7
FIGURE 7
Microalgae viability and renal tissue integrity after dynamic photosynthetic perfusion. Viability [(A), upper] and morphology [(A), lower] of the microalgae were not affected by the perfusion and the subsequent flushing step. H&E-stained paraffin sections, shows a normal histological structure of porcine kidneys in cortex [(B); top] and medulla [(B); bottom] after perfusion. Black and grey dots in A (bottom) indicate microalgae samples of the solution obtained before and after 10 min of perfusion, respectively, showing an almost complete overlapping of the signal where gray dots masked the microalgae population represented by black dots. Scale bar represents 200 µm [(B), left] and 30 µm [(B), right]. Data are expressed as mean ± SD; N = 2 in A.
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