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.
Green plants and algae synthesize adenosine triphosphate (ATP) and reducing nicotinamide adenine dinucleotide phosphate (NADPH), and excrete O2 by using light energy and H2O1. In the carbon-fixation cycle (Calvin cycle), glyceraldehyde-3-phosphate (G3P) is synthesized from CO2 and H2O by utilizing ATP and NADPH. Nutrients including carbohydrates are synthesized from G3P. On the other hand, animals including humans consume the O2 and nutrients in respiration and synthesize ATP. Carbon dioxide, produced by respiration, is reused as a core component in photosynthesis. In agriculture, animal excreta, manure, has been used as a valuable source of nitrogen for crop plants. Glutamate is synthesized from 2-oxoglutarate and ammonia, which is a metabolite of animals, by green plants and algae. Glutamine is synthesized from glutamate and ammonia. Other amino acids are synthesized by transamination. Animals use these amino acids. On the earth, all organisms are in a “symbiotic relation”, and the natural environment has built a “recycling society” by utilizing their carbon-nitrogen-oxygen cycles.
Primary cells isolated from biological tissues/organs can proliferate in vitro and are commonly utilized as cell sources in regenerative therapy. The number of cell-based regenerative and tissue-engineered therapies that can be clinically applied to repair damaged tissues/organs has rapidly expanded in the past few years2,3. Our laboratory has developed a temperature-responsive culture surface, and reported on the tissue engineering methodology, “cell sheet technology”, that makes use of this culture surface4. Because harvested cell sheets maintain their cell-cell junctions, cell surface proteins, and the extracellular matrix (ECM), cell-dense three-dimensional (3-D) tissues can be created by simply layering those cell sheets without any 3-D scaffolds to create tissues that engraft more efficiently onto target tissues without the need for suture5,6,7,8,9. The transplantation of cell sheets into various animal models with damaged tissues enabled the recovery of their original tissue functions, and numerous clinical studies using single- or multi-layered cell sheets have already been performed successfully7,8,9,10,11,12,13,14,15,16,17,18.
Recently, 3-D culture systems have become a focus in the field of cell biology19. The cellular environments of two-dimensionally (2-D) cultured cells are significantly different from the 3-D cultured cells, and these differences affect the gene expression and biochemical activity of those cells. Importantly, a 3-D culture system much more closely resembles in vivo situations20,21. A functional 3-D tissue, which surrogates actual living tissues, is also valuable as an in vitro tissue model to assess the efficacy and cytotoxicity of candidate drugs. An optimal 3-D tissue model can be expected in the fields of pharmacology and toxicology.
While cell-dense 3-D tissues can be easily created by the simple layering of cell sheets5,6, the ischemic environment makes the creation of thicker tissues difficult. The thickness limitation of 3-D tissues without vascular networks is approximately 40–80 μm22,23. Severe hypoxia/undernutrition within thicker multi-layered cell sheet-tissues without vascular networks is likely, which can induce tissue damage22,23,24. Thus, the thickness limitation of a viable tissue depends on O2/nutrient gradients. Moreover, noxious metabolites including lactate and ammonia have been reported to be toxic to cultured cells25. The accumulation can also induce cell death within thicker tissues. Cell death within thicker tissues can be controlled by supplying sufficient O2/nutrients and removing the metabolites. The creation of thicker tissues like organs is a long-standing goal in the field of tissue engineering, and the transplantation of thick tissues offers hope for more efficient therapies and the enlargement of the range of applications for regenerative therapies. Additionally, the thicker native-like tissue will be an optimal in vitro tissue model.
Here we report about the co-cultivation of mammalian cells and algae to simplify the creation of thicker tissue. This report shows that the co-cultivation method has some potential in the fields of cell biology, tissue engineering, and regenerative medicine.
Detection of O2 production from algae
An investigation was conducted to determine whether algae, Chlorococcum littorale26 (Fig. 1A), could be cultured and produce O2 within a culture media and temperature conditions optimized for mammalian cells. The algae were suspended in a medium for mammalian cells, and oxygen concentration in the medium was measured by an analysis system, which was developed recently27,28,29, shown schematically in Fig. 1B. Oxygen concentration was greater than the saturation concentration on the surface at 30 °C (Fig. 1C) and 37 °C (data not shown). The highest oxygen concentration was detected in the bottom of the culture dish, and the concentration decreased toward the surface, showing that the algae actively produced oxygen. However, after cultivation for 1 day at 37 °C, the algae showed a drastic decrease in oxygen production (data not shown), suggesting damage to the algae at such high temperatures. The algae, which were cultured at 37 °C for 5 days, showed a bleaching phenomenon (Fig. 1D). On the other hand, after cultivation at 30 °C for 1 day, the algae continued to produce oxygen, which was comparable with the precultivation (data not shown). Typically, many algae are cultured at 30 °C or less30, and healthy mammalian cells and cell sheets can also be functionally cultured at 30 °C (unpublished observation). Therefore, in the following experiments, the algae and mammalian cells were cultured at 30 °C (Table 1). Under dark conditions, oxygen concentration decreased to less than the concentration at the surface of the culture dish, namely, the saturated oxygen concentration (Fig. 1C), showing that the algae consumed oxygen under these conditions. It was suggested that the algae’s metabolism changed from photosynthesis to respiration by the absence of light. In this system, oxygen supply could be controlled by light.
Co-cultivation of mammalian cells with algae
Next, the oxygen consumption of mammalian cells was measured. In a culture dish without a C2C12 cell sheet, there was very little difference between the oxygen concentration at the surface and the bottom of the dish (Fig. 2A). However, in cultivation with a cell sheet the saturated oxygen concentration observed at the surface gradually decreased toward the bottom (Fig. 2A), showing that the confluent C2C12 cells actively consumed oxygen. When only the algae were present on the bottom without a C2C12 cell sheet, as shown in Fig. 1B (left), a high oxygen concentration (more than the saturated oxygen concentration level) was observed under light (Fig. 2B). However, the oxygen concentration under dark conditions was noted to be significantly lower (Fig. 2B). The result was similar to that of Fig. 1C. In the co-cultured area of the dish with a cell sheet and algae under light, shown schematically in Fig. 1B (right), the oxygen concentration in the bottom was similar to that on the surface (Fig. 2B), showing the algae actively produced oxygen, even in co-cultivation. Under dark conditions, the oxygen concentration in the co-culture area decreased significantly to the zero level (Fig. 2B). Under dark conditions, algae also consumed oxygen through respiration. Therefore, in the absence of light co-cultivation oxygen consumption was greater than the oxygen consumption in the cultivation of mammalian cells alone (Fig. 2A,B). In the same co-cultivation system of algae and another mammalian cell type, rat cardiac cells, similar oxygen profiles were also observed (data not shown). Under light, oxygen concentrations at the bottom of a C2C12 cell sheet or a rat cardiac cell sheet with algae were significantly higher than those at the bottom of same cell sheets alone (Fig. 2C). These results showed that the mammalian cells and the algae could be successfully co-cultured, and the algae were able to produce oxygen even under the co-cultivation conditions.
Co-cultivation of mammalian cells and algae for creating thicker tissues
To create thicker cardiac tissues, five or ten rat cardiac cell sheets were layered with or without algae as shown in Supplementary Fig. 1A, and the multi-cell layered tissues were then co-cultivated in a chamber under light conditions (Supplementary Fig. 1B). Firstly, cell metabolism within the tissues was examined. The glucose consumption and lactate production of a single-layer cardiac cell sheet hardly changed during co-cultivation (Fig. 3A,B). However, multi-layered cardiac cell sheets significantly decreased glucose consumption and lactate production in co-cultivation with algae (Fig. 3A,B). The ratio of lactate production to glucose consumption (L/G ratio) of cells was used as an index of the state of aerobic or anaerobic respiration. When all pyruvates, which are the final product of glycolysis, are converted to lactates in anaerobic respiration, the L/G ratio is two. On the other hand, in aerobic respiration pyruvate enters the mitochondrion to be fully oxidized by the tricarboxylic acid (TCA) cycle, and thus the L/G ratio becomes less than two. In the case of a single-layer cardiac cell sheet, the L/G ratio was 1.40 ± 0.07 (n = 3), showing partial aerobic respiration (Table 2). The L/G ratio [1.36 ± 0.07 (n = 3)] tended to be slightly lower in co-cultivation with algae (Table 2). In the single-layer cell sheet, because the tendency for aerobic respiration was so strong, the significant change of the L/G ratio may not have been detected due to the supply of oxygen from the algae. The L/G ratio of five- or ten-layered cardiac cell sheets was 1.82 ± 0.04 (n = 3) or 1.71 ± 0.14 (n = 5), respectively, showing that anaerobic respiration was higher within the thicker tissues (Table 2). However, the L/G ratios of those same five-layered or ten-layered cell sheets were decreased significantly to 1.41 ± 0.20 (n = 3) or 1.40 ± 0.14 (n = 5), respectively, in co-cultivation with algae, and the values were comparable with that of the single-layer cell sheet (Table 2). The result suggests that oxygenation within the multi-layered cell sheets occurred by the co-cultivation with algae as like the single-layer cell sheet, and a change from anaerobic respiration to partial aerobic respiration with the oxygen supply. Approximately 30 molar ATP are produced from the consumption of one molar glucose without lactate production in aerobic respiration, while only two molar ATP are produced by the consumption of one molar glucose in anaerobic respiration1. It was thought that the significant decrease in glucose consumption and lactate production of multi-layered cardiac cell sheet-tissues in co-cultivation with algae was related to effective ATP production in aerobic respiration.
Secondly, ammonia in the culture supernatant of multi-layered cardiac cell sheets was analyzed. Because the culture medium contained fetal bovine serum (FBS) and amino acids, including L-glutamine, ammonia was detected in the culture medium (Fig. 3C). The concentration of ammonia was increased by the cultivation of multi-layered cell sheets (Fig. 3C). However, ammonia within the culture medium of the multi-layered cardiac cell sheets was significantly lower [less than one-seventh (five-layered cell sheets) or one-eighth (ten-layered cell sheets)] in the co-cultivation with algae (Fig. 3C). Ammonia is produced by the degradation of amino acids. While ammonia is not used by animal cells, algae can use ammonia for producing amino acids. It was suggested that the algae used the ammonia within the culture medium to produce amino acids, resulting in the significant decrease in ammonia. Ammonia is an important source of nitrogen for the growth and survival of algae. Although algae can synthesize ammonia from atmospheric nitrogen through nitrogenase, eight molecules of ATP are used for the synthesis of one molecule of ammonia. Thus, the deficiency of ATP or the accumulation of adenosine diphosphate (ADP) inhibits nitrogenase activity. Ammonia is important for algae, so NH4Cl (50 μM) was supplied within the culture medium for the algae used in this study.
Thirdly, the condition of the cells within thicker cardiac tissues and the effect of the co-cultivation with algae were analyzed histologically. Five-layered cardiac cell sheets showed histologically damaged tissues with delamination of the cell sheets on the bottom, which was directly contacted with the dish (Fig. 4A). However, in the five-layered cell sheets with algae, viable stratified tissues containing algae were observed (Fig. 4B). Thicker cardiac tissues approximately 160 μm could be created by the co-culture system (Fig. 4B).
Fourth, creatine kinase (CK) release from the cardiac tissues was measured to detect any damage of the cardiomyocytes. The release of CK from cultured cells is used as a common index of damage and death of muscle cells including cardiomyocytes31. CK release from a single-layer cardiac cell sheet without algae or with algae was 5.7 ± 0.2 U or 3.7 ± 3.2 U, respectively (n = 3). In the ten-layered cardiac cell sheets without algae, the significant release of CK, which was more than 40-folds that of the single cell sheet, was detected (Fig. 5A), indicating severe damage of cardiomyocytes within the tissues. However, the amount released from the layered cell sheets with algae was decreased to less than one-fifth (Fig. 5A). Additionally, 80% of the ten-layered cardiac cell sheets without algae were partially or completely detached from the culture dishes as shown in Fig. 5B within 6 days of cultivation (4 of 5 cases), suggesting the severe tissue damage. However, all ten-layered cell sheets with algae were continuing to attach to the dishes for the follow-up periods (6 days) (n = 5, Fig. 5B). These results showed that the severe cell damage within the multi-layered cell sheets was relieved by the co-cultivation with algae.
On the earth, “symbiotic relationships” exist between various green plants, algae, animals, and bacteria, and together they build a“recycling society”. This study showed the possibility of an in vitro symbiotic relationship between mammalian cells and algae. A recycling system was created in which algae supplied O2 to mammalian cells and in turn reused the metabolic waste products (CO2, ammonia) from mammalian cells, while mammalian cells used the O2, and excreted CO2 and metabolites. The culture conditions within thicker multi-cell layered tissues were improved by this co-culture system. In the cultivation of thicker cell-dense tissues without algae, cell damage occurred within the tissues (Fig. 4), which in turn induced anaerobic respiration (Table 2). In anaerobic respiration, only two molar ATP per one molar glucose are produced and lactate is also produced1. Inefficient anaerobic respiration induced active glucose consumption and the active production of lactate (Fig. 3). The toxic effect of lactate on cells (pH and osmolarity) occurs at a concentration greater than 20–30 mM, and cell growth is reduced by ammonia concentrations greater than 2–3 mM25. In this study, the culture media of five-layered and ten-layered cardiac cell sheets contained approximately 5 mM lactate and 0.16 mM ammonia (data not shown). On the other hand, the diffusional inhibition of their molecules may induce high concentrated accumulation of lactate and ammonia just after the production from dense mammalian cells, and severe glucose deficiency and hypoxia at the microenvironment of the cells. This adverse environment induced severe cytotoxicity and tissue damage, especially at the bottom part, which made direct contact with the dish23 (Fig. 4). In the case of the co-cultivation of mammalian cells and algae, the algae produced O2 even in the conditions (Fig. 2B,C). The oxygen supply induced aerobic respiration even within the cell-dense tissue (Table 2). In aerobic respiration, approximately 30 molar ATP are produced by the consumption of one molar glucose and no lactate is produced1. The effective respiration induces mild glucose consumption and inhibits active lactate accumulation. Additionally, in the co-culture system, ammonia might be rapidly consumed by algae as a source of amino acid-synthesis just after their production. The co-cultivation showed a significant decrease in glucose consumption and the accumulation of lactate and ammonia (Fig. 3). Histological observation and the release of CK showed that the condition of the cells within the tissues was improved by the co-cultivation (Figs 4 and 5). The supply of O2 from algae and the consumption of ammonia by algae are thought to be closely related to the improvement of cell condition within thicker tissues. Thicker cardiac tissues approximately 160 μm could be created by the co-culture system, even though it has been reported that the thickness limitation of tissues without vascular networks is approximately 40–80 μm22,23. A native-like bioengineered tissue is useful as an in vitro surrogate tissue model for estimating the efficacy and cytotoxicity of candidate drugs. At present, we are preparing to estimate electrophysiologically and functionally whether a heterogeneous cardiac tissue with algae can be used as a tissue model for pharmacology and toxicology. On the other hand, the supply of nutrients/biological components (hydrocarbon, amino acids, and lipids) from algae to mammalian cells is as yet unproven. If the supply of components from algae to mammalian cells can be induced by some kind of method, for example, genetic recombination technology, ultimately mammalian cells may be able to be cultured in very poor solutions like phosphate-buffered saline. Algae have the ability to store/secrete energy-rich hydrocarbons32. At present, genetically engineered algae are being researched to efficiently obtain useful materials, for example, petroleum substitutes, bioethanol, and biodiesel32,33. Those techniques offer a possible opportunity for the efficient production and secretion of hydrocarbons, lipids, and amino acids. Advances in this area of research may induce to the fabrication of millimeter-level thicker organ-like tissues and low-cost cell culture, which would have a great impact on cell biology, medicine, and industry.
In the culture medium without cells, ammonia could be detected (Fig. 3C) because it contained FBS and all mammalian blood, including bovine, contains ammonia. Additionally, it is thought that the degradation of amino acids, especially L-glutamine, in the medium causes the detection of ammonia. The half-life of L-glutamine in media with serum (37 °C) is 3–25 days25. After 1 day-cultivation, approximately 0.13 mM ammonia in M199-based medium containing 6% FBS for cardiac cells and 0.39 mM in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS for C2C12 cells were detected (data not shown). The ammonia concentration may negatively affect growth, biochemical reactions and gene expression of cultured cells. The amount of ammonia in the culture medium was significantly reduced by the co-cultivation with algae (Fig. 3C). The development of a practical co-culture system with algae will have a positive impact on the normal culture of mammalian cells.
In this study, a co-cultivation system with algae was applied to create thicker bioengineered cardiac tissues. Cell sheet technology has applied to create and regenerate various tissues, and clinical studies have already been performed successfully in six different fields, including cardiovascular medicine, gastrointestinal medicine, ophthalmology, periodontal disease, orthopedic surgery, and otolaryngology10,12,13,14,15,16,17,18. Additional clinical research in the field of respiratory surgery with cell sheet technology is now being prepared34. It is thought that 109-level’s cells are necessary to treat just one patient with myocardial infarction or diabetes mellitus35. The creation of thicker healthy tissues allows us to transplant ever greater numbers of cells. The co-cultivation with algae allowed us to create multi-layered cell sheet-tissues (approximately 160 μm) at once under lights (Fig. 4). At present, we are preparing the transplantation of these multi-layered cell sheet-tissues into animal models. In some methods after transplantation, photoirradiation is necessary to maintain the thicker tissues in vivo until functional anastomoses with the host vasculature are formed. In a rat animal model, the functional anastomoses were rapidly formed within 12 hours after transplantation of cardiac cell sheet-tissues36. Because it was shown that the algae used in this study were damaged at 37 °C, the algae may also be damaged at the body temperature of a host. Our preliminary experiment showed that the algae did produce oxygen for several hours at 37 °C as like at 30 °C. On the other hand, some kinds of algae can be successfully cultured at 37 °C or even higher37. At present, we have started to co-culture thermostable algae and mammalian cells at 37 °C, and this system will contribute to the establishment of an optimal in vitro tissue-model as well as produce transplantable tissue. Inversely, the contamination of algae may have a problem after the formation of functional anastomoses with host vasculature. The thermosensitive algae used in this study could be removed gradually by the host body temperature so that only the target cells remain within the transplanted tissue. Moreover, at the time of tissue transplantation anti-algae antibody or the local application of non-toxic herbicides may be useful to be removed the algae from the tissue. Nevertheless, the contamination may be the cause of pathogenicity or immunogenicity after the transplantation. Recently, Schenck et al. reported about the transplantation of a bioartificial scaffold containing microalgae, Chlamydomonas reinhardtii, into a nude mouse full skin defect model or a zebrafish model38. In the experiment, no significant inflammatory response to the algae was observed in both mouse and zebrafish models. There are various kinds of algae. in vivo animal experiments, some algae will be selected as pathogenicity/immunogenicity-free algae. Concerning the in vivo transplantation of the heterogeneous tissue, more detailed investigation will be necessary.
This study proposed the possibility of an in vitro “symbiotic relationship” between mammalian cells and algae, and a “recycling system”, in which algae supply oxygen to mammalian cells, and reuse metabolites and waste products from mammalian cells; while mammalian cells consume the oxygen, and excrete metabolites and waste products. We believe that this new system has great potential for applications in the fields of cell biology, tissue engineering, and regenerative medicine.
All animal experiments were performed according to the Guidelines of Tokyo Women’s Medical University on Animal Use, and consistent with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources (ILAR). All the experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Women’s Medical University.
Cell culture and cell sheet preparation
C2C12 mouse myoblast lines (Sumitomo Dainippon Pharma, Osaka, Japan) were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Japan Bio Serum, Nagoya, Japan) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA), and C2C12 cell sheets were prepared according to previous reports5,6,24. Neonatal rat cardiac cells were isolated from the ventricles of 1-day-old Sprague-Dawley (SD) rats (CLEA, Tokyo, Japan), and cultured in an M199-based culture medium [40% Medium 199 (Invitrogen Corp., Carlsbad, CA, USA), 6% FBS, 0.2% penicillin-streptomycin, 2.7 mM glucose, and 54% balanced salt solution containing (in mM) 116 NaCl, 1.0 NaH2PO4, 0.8 MgSO4, 1.18 KCl, 0.87 CaCl2, and 26.2 NaHCO3], and a cardiac cell sheet was prepared as described in previous reports5,6,39,40,41.
Eukaryotic algae, Chlorococcum littorale, were obtained from the Biological Resource Center, National Institute of Technology and Evaluation (Tokyo, Japan), and were cultured in Daigo’s IMK Medium (Wako Pure Chemical, Tokyo, Japan) with synthetic seawater (Wako Pure Chemical) at room temperature (approximately 25–28 °C) under continuous light (approximately 500–700 lux). The algae (2.5 × 107 cells) were cultured in an M199-based culture medium at 30 °C or 37 °C under continuous light [1313 ± 45 lux (n = 3)] for 0 or 1 day on a 35-mm polystyrene culture dish (Corning, NY, USA), and then oxygen production was measured by an oxygen measurement system as described below. Illuminance was measured by illuminometer (As one, Osaka, Japan). Macroscopic photographs were taken by a digital camera (GR DIGITAL III, Ricoh, Tokyo, Japan).
Co-cultivation of cell sheets and algae, and histological analysis
The co-cultivation of mammalian cell sheets and the algae was performed as shown in Table 1. Single- or multi-layered cell sheet-tissues were cultured at 30 °C in a humidified atmosphere with 5% CO2 under continuous light [1313 ± 45 lux (n = 3)] in the culture box (Supplementary Fig. 1B). Culture media were changed every day, and harvested culture media were used for analyzing the metabolic activity and cell viability of the tissues. Layered cell sheets were fixed with 4% paraformaldehyde solution (Muto Pure Chemicals, Tokyo, Japan). Specimens were embedded in paraffin, sectioned, and subjected to a histological examination by hematoxylin-eosin staining. Prepared specimens were observed by an optical microscope (ECLIPSE E800) (Nikon, Tokyo, Japan). The images were processed using an imaging system (NIS-Elements) (Nikon).
The metabolic activities of cell sheet-tissues were monitored by measuring glucose, lactate, and ammonia concentrations in culture media. The cytotoxicity of cardiomyocytes was detected by CK release in culture media. The concentrations of glucose and lactate and CK activities were determined as described previously31,42,43,44. Total glucose consumption was calculated by subtracting the glucose concentration in the medium with cell sheets/algae or the cell sheets after the cultivation from the medium without cell sheets/algae after the cultivation. The values of lactate production and CK release were calculated by subtracting the backgrounds of their concentrations in the medium without cell sheets/algae or the cell sheets after the cultivation from the medium after the cultivation. Ammonia concentration was measured by a colorimetric method at an outsourcing laboratory, SRL (Tokyo, Japan).
Measurement of oxygen concentrations in culture medium
Recently, we have developed a measurement system for oxygen concentration using an oxygen microelectrode sensor [a Clark-type oxygen microsensor with a 8–12 μm-diameter tip made of fragile glass (OX-10) (Unisense, Denmark)] and a high-precision electronic balance (HTR-220) (Shinko Denshi, Tokyo, Japan), which can detect the position of the tip of the oxygen sensor when it comes into contact with the bottom of the dish27,28,29. The measurement was performed in a humidified atmosphere containing 20% oxygen and 5% CO2 in a glove box hypoxia workstation (INVIVO2 300) (Ruskinn Technology, Mid Glamorgan, UK). Oxygen concentrations in culture dishes were estimated by the system with or without light [1103 ± 25 lux (n = 2)]. The measurements in Figs 1C and 2A,B were performed at three points, and the two independent experiments were also performed. The representable profiles were shown in the figures.
Data in Figs 2C, 3 and 5A are expressed as means ± standard deviation. An unpaired Student t-test was performed to compare two groups. One-way analysis of variance (Ryan’s method) was used for multiple group comparisons. A p-value of less than 0.05 was considered significant.
How to cite this article: Haraguchi, Y. et al. Thicker three-dimensional tissue from a “symbiotic recycling system” combining mammalian cells and algae. Sci. Rep. 7, 41594; doi: 10.1038/srep41594 (2017).
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We appreciate the useful comments and technical criticism from Dr. Hiroshi Sekiguchi and Mr. Kazuya Okada (Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan). We also thank Dr. Escobedo-Lucea Carmen and Mr. Allan Nisbet for their useful comments and editing assistance. This work was supported by a grant from the Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and from CellSeed Inc.
There is potential competing interest. Teruo Okano is a founder and a member of the board of CellSeed Inc., which has licenses for certain cell sheet-related technologies and patents from Tokyo Women’s Medical University. Tatsuya Shimizu and Teruo Okano are shareholders of CellSeed Inc. Tokyo Women’s Medical University is receiving research funds from CellSheed Inc.
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Haraguchi, Y., Kagawa, Y., Sakaguchi, K. et al. Thicker three-dimensional tissue from a “symbiotic recycling system” combining mammalian cells and algae. Sci Rep 7, 41594 (2017). https://doi.org/10.1038/srep41594
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