Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production.

Muscle cell cultivation, specifically the culture of artificial meat from livestock-derived cells in serum-free media is an emerging technology and has attracted much attention. However, till now, the high cost of production and environmental load have been significant deterrents. This study aims to provide an alternate growth-promoting substance that is free from animal derivatives and lowers nitrogen pollution. We have extracted water-soluble compounds from the filamentous nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 by the ultrasonication method. The heat-inactivated and molecular weight separation experiments were conducted to identify the bioactive compound present in the extract. Finally, the compounds soluble in water (CW) containing the water-soluble pigment protein, phycocyanin as a bioactive compound, was added as a growth supplement to cultivate muscle cells such as C2C12 muscle cells and quail muscle clone 7 (QM7) cells to analyze the effectiveness of the extract. The results indicated that CW had a positive role in muscle cell proliferation. A three-dimensional (3-D) cell-dense structure was fabricated by culturing QM7 cells using the extract. Furthermore, the nitrogen-fixing cyanobacterial extract has vast potential for cultured meat production without animal sera in the near future.

Circular cell culture for sustainable food production using recombinant lactate-assimilating cyanobacteria that supplies pyruvate and amino acids.

Recently, we reported a circular cell culture (CCC) system using microalgae and animal muscle cells for sustainable culture food production. However, lactate accumulation excreted by animal cells in the system characterized by medium reuse was a huge problem. To solve the problem, as an advanced CCC, we used a lactate-assimilating cyanobacterium Synechococcus sp. PCC 7002, using gene-recombination technology that synthesises pyruvate from lactate. We found that the cyanobacteria and animal cells mutually exchanged substances via their waste media: (i) cyanobacteria used lactate and ammonia excreted by animal muscle cells, and (ii) the animal cells used pyruvate and some amino acids excreted by the cyanobacteria. Because of this, animal muscle C2C12 cells were amplified efficiently without animal serum in cyanobacterial culture waste medium in two cycles (first cycle: 3.6-fold; second cycle: 3.9-fold/three days-cultivation) using the same reuse medium. We believe that this advanced CCC system will solve the problem of lactate accumulation in cell culture and lead to efficient cultured food production.

A circular cell culture system using microalgae and mammalian myoblasts for the production of sustainable cultured meat.

For sustainable production of cultured meat, we propose a novel circular cell culture (CCC) system in which microalgae are used as nutrient supply for the mammalian cell culture and as a waste-medium recycler. Chlorococcum littorale, RL34 hepatocytes, and C2C12 myoblasts were used as cell sources for microalgae, growth factor-producing cells, and muscle cells, respectively. In the first cycle, C2C12 cells were amplified 4.0-fold after 48 h of culture in an RL34 cell-conditioned medium. In the second cycle, C2C12 cells were cultured in the C. littorale culture waste medium to which the C. littorale-derived nutrients were added. The proliferation rates of C. littorale and C2C12 and the nutrient extraction efficiency from C. littorale were the same in the first and second cycles. Therefore, this CCC system, which works without additional grain-derived nutrients and animal sera, will help drastically reduce environmental load, resource/energy consumption, and costs in future cultured meat production.

Microalgal culture in animal cell waste medium for sustainable 'cultured food' production.

'Cultured food' has tremendous potential as a sustainable meat alternative. Increased cultured food production is increasing the amount of waste medium from cell culture. Nitrogen- and phosphorus-containing compounds in waste medium can cause eutrophication of water bodies. Currently, microalgae are used in energy production, environmental protection, agriculture and pharmaceutical and health food industries. Here, we used the microalgae, Chlorococcum littorale and Chlorella vulgaris and the waste medium of C2C12 cells for a case study. We found that 80% and 26% of ammonia and 16% and 15% of phosphorus in the waste medium were consumed by C. littorale and C. vulgaris, respectively. In addition, C. littorale and C. vulgaris proliferated 3.2 folds and 1.6 folds, respectively, after seven days in the waste medium that was enhanced by adjusting medium salt concentration. This report demonstrates the potential of sustainability for solving the issue of waste medium production during the production of cultured food.

Three-dimensional tissue fabrication system by co-culture of microalgae and animal cells for production of thicker and healthy cultured food.

OBJECTIVES: "Cultured food" is focused worldwide as "the third stage in meat production system" after hunting and livestock farming, and a sustainable food production system. In this study, we attempted to fabricate a three-dimensional (3-D) tissue by co-cultivation of animal cells with photosynthetic autotrophic microalgae so as to produce thicker and healthy cultured foods. RESULTS: Metabolism and damage of co-cultured tissues fabricated by microalgae, Chlorella vulgaris (C. vulgaris), and C2C12 cells were compared to monoculture tissues fabricated by C2C12 animal cells alone. Although the metabolism of monoculture tissue showed anaerobic respiration (ratio of lactate production to glucose consumption, LG ratio: 2.01 ± 0.15), that of the co-culture tissue partially changed to efficient aerobic respiration (LG ratio: 1.58 ± 0.14). In addition, the amount of ammonia in the culture media decreased markedly by co-cultivation. The release of lactate dehydrogenase from the thicker tissue was one-seventh in the co-cultivation, showing improved tissue damage. The co-cultivation with microalgae improved the culture condition of thicker tissues, resulting in the fabrication/maintenance of 200-400 µm-thickness tissues. The co-cultured tissue fabricated by microalgae and animal cells was not only rich in nutrients but also enabled thicker tissue fabrication without tissue damage as compared to tissue fabricated by animal cells alone. CONCLUSIONS: This tissue fabrication system by co-culture of microalgae and animal cells will be a valuable tool for the production of thicker and healthy cultured food.

In situ cross-linked electrospun fiber scaffold of collagen for fabricating cell-dense muscle tissue.

Engineered muscle tissues used as transplant tissues in regenerative medicine should have a three-dimensional and cell-dense structure like native tissue. For fabricating a 3D cell-dense muscle tissue from myoblasts, we proposed the electrospun type I collagen microfiber scaffold of the string-shape like a harp. The microfibers were oriented in the same direction to allow the myoblasts to align, and were strung at low density with micrometer intervals to create space for the cells to occupy. To realize this shape of the scaffold, we employed in situ cross-linking during electrospinning process for the first time to collagen fibers. The collagen microfibers in situ cross-linked with glutaraldehyde stably existed in the aqueous media and completely retained the original shape to save the spaces between the fibers for over 14 days. On the contrary, the conventional cross-linking method by exposure to a glutaraldehyde aqueous solution vapor partially dissolved and damaged the fiber to lose a low-density shape of the scaffold. Myoblasts could penetrate into the interior of the in situ cross-linked string-shaped scaffold and form the cell-dense muscle tissues. Histochemical analysis showed the total area occupied by the cells in the cross section of the tissue was approximately 73 %. Furthermore, the resulting muscle tissue fabricated from primary myoblasts showed typical sarcomeric cross-striations and the entire tissue continuously pulsated by autonomous contraction. Together with the in situ cross-linking, the string-shaped scaffold provides an efficient methodology to fabricate a cell-dense 3D muscle tissue, which could be applied in regenerative medicine in future.

Oxygen consumption of human heart cells in monolayer culture.

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71±0.38pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77±0.32pmol/h/cell and 1.61±0.70pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Chondrocyte differentiation of human endometrial gland-derived MSCs in layered cell sheets.

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy.

Development of serum-free and grain-derived-nutrient-free medium using microalga-derived nutrients and mammalian cell-secreted growth factors for sustainable cultured meat production.

Considering the amount of global resources and energy consumed, and animal welfare issues associated with traditional meat production, cultured meat production has been proposed as a solution to these problems and is attracting worldwide attention. Cultured meat is produced by culturing/proliferating animal muscle cells in vitro. This process requires significant amounts of culture medium, which accounts to a major portion of the production cost. Furthermore, it is composed of nutrients derived from grains and heterotrophic microorganisms and fetal bovine serum (FBS), which will impact the sustainability of cultured meat in future. Here, we developed a novel medium containing nutrients extracted from microalga and cell-secreted growth factors. First, rat liver epithelial RL34 cells were cultured by adding Chlorella vulgaris extract (CVE) to inorganic salt solution. The supernatant, containing the RL34 cell-secreted growth factors, was used as the conditioned medium (CM). This CM, with CVE added as a nutrient source, was applied to primary bovine myoblast cultures. This serum-free and grain-derived-nutrient-free medium promoted the proliferation of bovine myoblasts, the main cell source for cultured beef. Our findings will allow us to take a major step toward reducing production costs and environmental impacts, leading to an expansion of the cultured meat market.

L-Lactate treatment by photosynthetic cyanobacteria expressing heterogeneous L-lactate dehydrogenase.

L-Lactate is a major waste compound in cultured animal cells. To develop a sustainable animal cell culture system, we aimed to study the consumption of L-lactate using a photosynthetic microorganism. As genes involved in L-lactate utilization were not found in most cyanobacteria and microalgae, we introduced the NAD-independent L-lactate dehydrogenase gene from Escherichia coli (lldD) into Synechococcus sp. PCC 7002. The lldD-expressing strain consumed L-lactate added to basal medium. This consumption was accelerated by expression of a lactate permease gene from E. coli (lldP) and an increase in culture temperature. Intracellular levels of acetyl-CoA, citrate, 2-oxoglutarate, succinate, and malate, and extracellular levels of 2-oxoglutarate, succinate, and malate, increased during L-lactate utilization, suggesting that the metabolic flux from L-lactate was distributed toward the tricarboxylic acid cycle. This study provides a perspective on L-lactate treatment by photosynthetic microorganisms, which would increase the feasibility of animal cell culture industries.

Creation of human cardiac cell sheets using pluripotent stem cells.

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat.

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.

Production of scaffold-free cell-based meat using cell sheet technology.

In the production of cell-based meat, it is desirable to reduce animal-derived materials as much as possible to meet the challenges of sustainability. Here, we demonstrate the "cell sheet-based meat": scaffold-free cell-based meat using cell sheet technology and characterize its texture and nutrients. Bovine myoblast cell sheets were prepared using temperature-responsive culture dishes (TRCDs) and 10 stacked cell sheets to fabricate three-dimensional tissue of 1.3-2.7 mm thickness. Hardness was increased by incubation on the TRCD and was further increased by boiling as is characteristic of natural meat. The wet weight percentage of total protein in the cell sheet was about half that of beef. In this method, large-sized items of cell sheet-based meat were also created by simply scaling up the TRCD. This method promises an environment-friendly food product.

Mammalian cell cultivation using nutrients extracted from microalgae.

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of "cultured meat." Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes.

Analysis of force vector field during centrifugation for optimizing cell sheet adhesion.

A three-dimensional tissue was fabricated by layering cell sheets with centrifugation. In this system, an optimal centrifugal force promoted the adhesion between (a) a cell sheet and a culture dish, and (b) layered cell sheets, resulting in a significant decrease in the fabrication time of the tissue. However, negative effects like sliding/significant deformation of cell sheets were observed upon high rotational speed use. These negative effects inhibit the further shortening of the fabrication time. The sliding/deformation suggests that the centrifugal forces were applied on the cell sheets in unwanted directions. Studies on the force vector field applied to the object placed on the plate during centrifugation are not available, and thus, the reason for the occurrence of such negative effects is unclear. Here, we theoretically derived the spatial distribution of acceleration applied on a plate during centrifugation. Using this theory, we found that the negative effects were triggered by the centrifugal force in the direction parallel to the plate surface, which appeared due to an inclination of the plate surface against a horizontal plane. Therefore, by adding weights on the plate edge to maintain the plate surface in a horizontal position, we succeeded in eliminating the negative effects and in increasing the rotational speed, with the minimum risk of sliding/deformation of cell sheets. We succeeded in reducing the time to establish tight adhesion between a mouse myoblast sheet and a culture dish, and layered cell sheets by increasing the centrifugal force from 5 min to 1 min without significant cytotoxicity.

Rapid creation system of morphologically and functionally communicative three-dimensional cell-dense tissue by centrifugation.

This study reports a rapid fabrication system of a morphologically and functionally communicative three-dimensional (3D) cell-dense tissue without scaffolds by centrifugation. The tight adhesion between C2C12 myoblasts and culture surface was accelerated without significant cell damage by centrifugation (80 x g, 37 °C, 30 min). A thicker tissue created on a temperature-responsive culture surface was harvested by decreasing temperature. The 3D myoblast tissues having approximately 200 µm-thickness were created at 1.5 h [centrifugation (80 x g, 37 °C) for 30 min and tissue harvest for 1 h]. However, in the case of without centrifugation, the myoblast tissues had fragile parts even at 7.5 h after the incubation. Additionally, electrically/functionally communicative and thicker human induced pluripotent stem (iPS) cell-derived cardiac tissues were created rapidly by the centrifugation and cultivation at 37 °C. We report a centrifugation system that significantly shortens the creation time of 3D tissues. We envision that this procedure will contribute to the field of tissue engineering and regenerative medicine. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1447-1453, 2018.

Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge.

Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:692-701, 2018.

Contractile force measurement of human induced pluripotent stem cell-derived cardiac cell sheet-tissue.

We have developed our original tissue engineering technology "cell sheet engineering" utilizing temperature-responsive culture dishes. The cells are confluently grown on a temperature-responsive culture dish and can be harvested as a cell sheet by lowering temperature without enzymatic digestion. Cell sheets are high-cell-density tissues similar to actual living tissues, maintaining their structure and function. Based on this "cell sheet engineering", we are trying to create functional cardiac tissues from human induced pluripotent stem cells, for regenerative therapy and in vitro drug testing. Toward this purpose, it is necessary to evaluate the contractility of engineered cardiac cell sheets. Therefore, in the present study, we developed a contractile force measurement system and evaluated the contractility of human iPSC-derived cardiac cell sheet-tissues. By attaching the cardiac cell sheets on fibrin gel sheets, we created dynamically beating cardiac cell sheet-tissues. They were mounted to the force measurement system and the contractile force was measured stably and clearly. The absolute values of contractile force were around 1 mN, and the mean force value per cross-sectional area was 3.3 mN/mm2. These values are equivalent to or larger than many previously reported values, indicating the functionality of our engineered cardiac cell sheets. We also confirmed that both the contractile force and beating rate were significantly increased by the administration of adrenaline, which are the physiologically relevant responses for cardiac tissues. In conclusion, the force measurement system developed in the present study is valuable for the evaluation of engineered cardiac cell sheet-tissues, and for in vitro drug testing as well.

Real-time quantitation of internal metabolic activity of three-dimensional engineered tissues using an oxygen microelectrode and optical coherence tomography.

Recent progress in tissue engineering technology has enabled us to develop thick tissue constructs that can then be transplanted in regenerative therapies. In clinical situations, it is vital that the engineered tissues to be implanted are safe and functional before use. However, there is currently a limited number of studies on real-time quality evaluation of thick living tissue constructs. Here we developed a system for quantifying the internal activities of engineered tissues, from which we can evaluate its quality in real-time. The evaluation was achieved by measuring oxygen concentration profiles made along the vertical axis and the thickness of the tissues estimated from cross-sectional images obtained noninvasively by an optical coherence tomography system. Using our novel system, we obtained (i) oxygen concentration just above the tissues, (ii) gradient of oxygen along vertical axis formed above the tissues within culture medium, and (iii) gradient of oxygen formed within the tissues in real-time. Investigating whether these three parameters could be used to evaluate engineered tissues during culturing, we found that only the third parameter was a good candidate. This implies that the activity of living engineered tissues can be monitored in real-time by measuring the oxygen gradient within the tissues. The proposed measuring strategy can be applied to developing more efficient culturing methods to support the fabrication of engineered thick tissues, as well as providing methods to confirm the quality in real-time. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 855-864, 2017.