Thicker three-dimensional tissue from a "symbiotic recycling system" combining mammalian cells and algae

Abstract

In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac cell-layered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 μm-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro "symbiotic recycling system" composed of mammalian cells and algae.

Figure 1. Oxygen measurement system and algae, Chlorococcum littorale.

A photograph of the algae (A). The schematic illustration of the system for oxygen concentration measurement (B). Representative oxygen concentration profiles plotted against the height from the bottom of the dish used for culturing the algae in an M199-based culture medium with/without light at 30 °C (C). The photographs of the algae after 0-day- (left) and 5-day-cultivation (right) at 37 °C on 35-mm polystyrene culture dishes (D).

Figure 2. Cultivation of single-layer C2C12 cell sheet or rat cardiac cell sheet with/without algae, Chlorococcum littorale.

Representative oxygen concentration profiles plotted against the height from the bottom of a culture dish used for culturing a C2C12 cell sheet without (A) and with the algae (B). In (B) panel the experiments were performed with/without light. The dissolved oxygen concentrations at the bottom of the dish, in which a C2C12 cell sheet or a rat cardiac cell sheet was cultured with/without the algae, are the average of the obtained data, which were the average values from the three points measured (n = 2) (C). *p < 0.05.

Figure 3. Cell metabolism of single- or multi-layered rat cardiac cell sheets with/without algae, Chlorococcum littorale.

Comparisons of glucose consumption (A) and lactate production (B), and ammonia amounts (C) in the medium for 24 h-cultivation by single- or multi-layered cardiac cell sheets with/without the algae. Single- and five-layered cell sheets: n = 3; ten-layered cell sheets: n = 5. *p < 0.05.

Figure 4. Histological observation of multi-layered rat cardiac cell sheets with/without algae, Chlorococcum littorale.

The photographs are a histological observation with hematoxylin-eosin staining of five-layered cardiac cell sheets without (A) or with (B) the algae after a 3-day cultivation, respectively. Three independent experiments were performed and all the experiments showed similar results. The representative photographs are shown in the figure.

Figure 5. Cytotoxicity assessment of multi-layered rat cardiac cell sheets with/without algae, Chlorococcum littorale.

The total creatine kinase (CK) release from ten-layered rat cardiac cell sheets with/without the algae during 4 days of cultivation is shown (A, n = 5). The release of CK was reduced to less than one-fifth by cocultivation with algae. *p < 0.05. Photographs of ten-layered rat cardiac cell sheets without (left) and with the algae (right) on 60-mm polystyrene culture dishes (B).