TY - JOUR
AU - Ushiki,, Tatsuo
AB - Abstract Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues. scanning electron microscopy (SEM), low-melting-point agarose, transmission electron microscopy (TEM), osmium-maceration method, free cells, culture cells Introduction Scanning electron microscopy (SEM) yields information about the surface topography of specimens and has been widely used in biomedical fields for analyzing the three-dimensional (3D) surface structure of cells and tissues. Most of these studies are concerned with the 3D cytoarchitecture of tissues and organs as well as the cell surface morphology. Tanaka and Naguro [1] and Tanaka and Mitsushima [2] have introduced osmium-maceration methods for SEM of intracellular organelles. The osmium-maceration method uses a diluted OsO4 solution for removing cytoplasmic soluble proteins from the fractured surface of the fixed cells, which enable 3D visualization of membranous cell organelles by high-resolution SEM. Using these techniques, cellular structures such as mitochondria, endoplasmic reticulum (ER) and Golgi apparatus have been observed three-dimensionally in different kinds of cells [3–8]. Recently, we have also demonstrated the 3D ultrastructure of the Golgi apparatus in different cells, that is, epididymal principal cells, goblet cells, gonadotrophs and dorsal root ganglion cells in the rat, by high-resolution SEM of osmium-macerated tissues [9]. Although most of these studies using the maceration methods have been performed on fractured tissue blocks, only a few studies of free or cultured cells have used these maceration methods. This is because free and cultured cells should be embedded in materials to produce a fractured surface before osmium maceration. The embedding materials should be resistant to osmium treatment should not penetrate the cells. For this purpose, a chitosan-embedding method was previously introduced by Fukudome and Tanaka [10]. However, this method has not been widely accepted because it is rather complicated and time-consuming. In contrast, Isola et al. [11] have recently succeeded in observing the 3D ultrastructure of Candida albicans by high-resolution SEM after embedding the yeast in low-melting-point agarose, followed by osmium maceration. This study gave us the idea that agarose might be a useful embedding material for studying not only microorganisms, but also free or cultured cells by SEM after osmium maceration. Thus, we used agarose embedding of free and cultured cells for SEM observation of their organelles, using the osmium-maceration method. Agarose-embedded samples can also be observed by light microscopy (LM) and transmission electron microscopy (TEM), and therefore, we used this method for 3D analysis of the intracellular structure of cultured cells by SEM, LM and TEM. Materials and methods HeLa cells were kindly provided by Dr Toru Hirota, Department of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan, and human leukocytes were collected from the peripheral blood which was obtained from a healthy male volunteer who gave informed consent. Blood collection was approved by the Ethics Committee of Niigata University School of Medicine. HeLa cells were grown on culture dishes (50 mm in diameter) for 48 h in a CO2 incubator at 38°C until the cells became confluent (cell count ∼2 × 104 µl−1). The dishes are commercial, temperature-responsive culture dishes (Up Cell, Cell Seed, Tokyo, Japan), which are useful for harvesting cultured cells because the cells can be detached from the dish by reducing the temperature below 20°C. After cultivation, the dishes were placed at 20°C for 20 min, the medium was replaced with phosphate buffered saline (PBS) and cells were suspended using a pipette. The cell suspension was centrifuged at 330 × g for 5 min so as to collect the cells as pellets. After removal of the supernatant, the pellet was fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB at pH 7.4 for 30 min at 4°C. The suspension was then centrifuged at 330 × g for 5 min to collect the cell pellets. For preparing leukocytes, 20 ml of peripheral blood was collected with a heparin-coated syringe from the basilic vein of a donor. Heparinized peripheral blood was diluted at a ratio of 1:1 with PBS and each 10 ml of diluted blood sample was carefully layered on a 4-ml Ficoll-Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) medium in a 15-ml centrifuge tube. The tubes were centrifuged at 400 × g for 30 min and leukocytes were gently collected by a Pasteur pipette from the cell layer and then transferred to a clean tube. The samples were centrifuged at 330 × g for 10 min in order to collect the cell pellets (cell count ∼3 × 104 µl−1). After removing the supernatant, the pellets were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB at pH 7.4 for 30 min at 4°C. The suspension was centrifuged at 330 × g for 10 min to collect the leukocyte pellets. The HeLa cells and leukocytes were rinsed in 0.1 M PB for 10 min and centrifuged at 330 × g for 5 min. After removal of the supernatant, the cell pellets were further fixed with 1% osmium tetroxide (OsO4) in 0.1 M PB for 30 min. They were centrifuged at 330 × g for 5 min, and rinsed with the same buffer for 10 min after removal of the supernatant, and centrifuged again at 330 × g for 5 min. The pellets were then suspended in 1 ml of 0.1 M PB (Fig. 1 [1]) and mixed with 3 ml of 5% low-melting-point agarose solution (Sigma, St Louis, MO), which was previously melted and cooled in a 35-mm diameter Petri dish to maintain the temperature between 35 and 40°C (Fig. 1 [2]). The mixing ratio between the cell suspension and agarose medium was ∼1:3. The mixture of cells and agarose in the Petri dish was gently and carefully stirred with the tip of a pipette to avoid formation of air bubbles (Fig. 1 [3]), and the dish was placed in a refrigerator for 5 min to harden the agarose by cooling (Fig. 1 [4]). The chilled agarose was peeled off from the Petri dish, placed on a wax sheet (Paraffin Wax, GC, Tokyo, Japan) and then cut into small blocks (1 × 1 × 1 mm for TEM; 2 × 2 × 5 mm for SEM) with razor blades (Fig. 1 [5–8]). Fig. 1. Open in new tabDownload slide Schematic drawing of the agarose-embedding method. [1] Cell suspension (C). [2] Cell suspension (C) is mixed with 5% low-melting-point agarose (a) in a Petri dish. [3] Agarose with cell suspension is stirred gently to avoid formation of air bubbles. [4] Agarose in Petri dish is cooled in a refrigerator for hardening. [5] Gelled agarose is peeled off from a Petri dish. [6–8] Agarose is cut into small blocks (TEM: 1 mm × 1 mm × 1 mm; SEM: 2 mm × 2 mm × 5 mm) with razor blades. Fig. 1. Open in new tabDownload slide Schematic drawing of the agarose-embedding method. [1] Cell suspension (C). [2] Cell suspension (C) is mixed with 5% low-melting-point agarose (a) in a Petri dish. [3] Agarose with cell suspension is stirred gently to avoid formation of air bubbles. [4] Agarose in Petri dish is cooled in a refrigerator for hardening. [5] Gelled agarose is peeled off from a Petri dish. [6–8] Agarose is cut into small blocks (TEM: 1 mm × 1 mm × 1 mm; SEM: 2 mm × 2 mm × 5 mm) with razor blades. LM and TEM Small specimen blocks were dehydrated in graded ethanol and embedded in epoxy resin. For LM, semi-thin sections (1 µm thick) were cut using an ultramicrotome (Leica Microsystems, Nussloch, Germany), stained with toluidine blue and observed with a conventional LM. For TEM, ultrathin sections of 100 nm thickness were cut using the ultramicrotome and mounted on grids. The sections were contrasted with aqueous solution of uranyl acetate and lead citrate, and examined in a Hitachi H-7650 TEM (Tokyo, Japan) at an accelerating voltage of 80 kV. SEM of osmium-macerated agarose blocks The agarose blocks were immersed in 25% and 50% dimethyl sulfoxide (DMSO) in distilled water for 30 min each. They were then frozen on a flat aluminum block that had been pre-cooled with liquid nitrogen in a vacuum bottle, and broken into two pieces with a screwdriver and a hammer on the block. The fractured pieces were immediately replaced in 50% DMSO for thawing at room temperature, and rinsed in 0.1 M PB (10 min, five times) until the DMSO had been completely removed. For cell maceration, specimens were immersed in 0.1% OsO4 in 0.1 M PB for 48 h at 20–22°C. The macerated specimens were further fixed with 1% OsO4 in 0.1 M PB for 1 h, and rinsed in a buffer solution for 1 h. They were then conductive stained by treating with 1% tannic acid (Nacalai Tesque, Kyoto, Japan) in 0.1 M PB for 1 h, rinsing in the same buffer for 1 h and immersing in 1% OsO4 in 0.1 M PB for 1 h [12]. The samples were dehydrated through a graded ethanol series, transferred to isoamyl acetate and dried in a critical point dryer (HCP-2, Hitachi, Tokyo, Japan) with liquid CO2. After the fractured surface was checked under a dissecting microscope, dried specimens were mounted on aluminum stubs with silver paste, and coated lightly (<5 nm) with platinum and palladium in an ion-sputter coater (E1010, Hitachi, Tokyo, Japan). They were observed in a Hitachi S-5000 in-lens-type field emission SEM (Tokyo, Japan) at an accelerating voltage of 5 kV. Results LM and TEM of HeLa cells HeLa cells embedded in agarose were observed by LM of semi-thin sections (Fig. 2a). LM showed that HeLa cells were effectively harvested in agarose at high density. Epon-embedded agarose was transparent and was not stained with toluidine blue and, therefore, cells were observed as if they were directly embedded in epoxy resin. In ultrathin sections for TEM, agarose was also transparent (Fig. 2b), whereas the ultrastructure of the cells including Golgi apparatus, mitochondria, rough ER and centrioles were well preserved and clearly observed by higher magnification of TEM images (Fig. 2c and d). The Golgi apparatus consists of small stacks of Golgi cisterns, which were piled up in layers, and small vesicles were observed around the Golgi. The mitochondria were roughly spherical, and dilated or tubular cisterns of rough ER were scattered throughout the cytoplasm. Fig. 2. Open in new tabDownload slide LM and TEM of agarose-embedded HeLa cells. (a) Semi-thin sections of agarose-embedded HeLa cells, which are stained with toluidine blue. This LM shows that HeLa cells were effectively harvested in agarose, whereas agarose was transparent between cells (filled star). (b) Low-magnification TEM image of an agarose-embedded HeLa cell. Agarose was transparent even in this micrograph (filled star). N, nucleus. (c and d) Higher magnification image of the HeLa cell. The Golgi apparatus (G), rough ER (rER), mitochondria (M) and a centriole (Ce) were observed in the cytoplasm. N, nucleus. This figure is available in black and white in print and in colour at JEM online. Fig. 2. Open in new tabDownload slide LM and TEM of agarose-embedded HeLa cells. (a) Semi-thin sections of agarose-embedded HeLa cells, which are stained with toluidine blue. This LM shows that HeLa cells were effectively harvested in agarose, whereas agarose was transparent between cells (filled star). (b) Low-magnification TEM image of an agarose-embedded HeLa cell. Agarose was transparent even in this micrograph (filled star). N, nucleus. (c and d) Higher magnification image of the HeLa cell. The Golgi apparatus (G), rough ER (rER), mitochondria (M) and a centriole (Ce) were observed in the cytoplasm. N, nucleus. This figure is available in black and white in print and in colour at JEM online. SEM of HeLa cells and human blood cells The fractured surface of the agarose blocks after osmium maceration was clearly identified because of its smoothness under the dissecting microscope. When the surface was observed by SEM, cells embedded in agarose were fractured together with agarose. SEM images also showed that agarose was not dissolved during osmium maceration and it filled the space between the cells (Fig. 3a and b). At high magnification, the fractured surface of agarose was observed as dense spongy structures. These structures filled the small gaps between the microvillous projections on the outer cell surfaces, but did not penetrate the cells. In contrast, membranous cell organelles such as mitochondria, Golgi apparatus and rough ER were well preserved in the fractured surface of the osmium-macerated cells. In HeLa cells, mitochondria were round and had plate-like cristae within the mitochondrial matrix (Fig. 3c). The small Golgi apparatus was located in the juxtanuclear region and had Golgi cisterns piled up in layers. A large number of vesicles were present around the Golgi apparatus. Rough ER was densely packed throughout the cytoplasm. Fig. 3. Open in new tabDownload slide Scanning electron micrographs of agarose-embedded and osmium-macerated HeLa cells. (a) Fractured surface of the agarose block, where HeLa cells were also fractured together with agarose. HeLa cells were surrounded by the agarose medium (filled star). Cell nuclei are colored purple with the Photoshop graphic software. (b) Low-magnification image of a HeLa cell. The cytoplasm (cyan) of the HeLa cell is occupied with membranous cell organelles. The boxed area with a white broken line is shown in (c). Purple, nucleus. Filled star, agarose medium. (c) Closer view of a part of (b). Many vesicles are present around the Golgi apparatus (blue). The mitochondria (green) are round, and plate-like cristae are seen within the matrix. Yellow, rough ER; red, lysosomes; purple, nucleus. Fig. 3. Open in new tabDownload slide Scanning electron micrographs of agarose-embedded and osmium-macerated HeLa cells. (a) Fractured surface of the agarose block, where HeLa cells were also fractured together with agarose. HeLa cells were surrounded by the agarose medium (filled star). Cell nuclei are colored purple with the Photoshop graphic software. (b) Low-magnification image of a HeLa cell. The cytoplasm (cyan) of the HeLa cell is occupied with membranous cell organelles. The boxed area with a white broken line is shown in (c). Purple, nucleus. Filled star, agarose medium. (c) Closer view of a part of (b). Many vesicles are present around the Golgi apparatus (blue). The mitochondria (green) are round, and plate-like cristae are seen within the matrix. Yellow, rough ER; red, lysosomes; purple, nucleus. The 3D structure of the cell organelles was also observed in human leukocytes by SEM (Fig. 4). Lymphocytes were small (about 5 µm in diameter), round and had a spherical nucleus that was surrounded by a thin layer of cytoplasm (Fig. 4a). Neutrophils were easily distinguished by their lobulated nucleus (Fig. 4b). Small stacks of Golgi apparatus and a few mitochondria were present within the cytoplasm (Fig. 4c). A large number of the specific and azurophilic granules were observed throughout the cytoplasm. Higher magnification images showed that the fine structure of the Golgi apparatus and mitochondria were well preserved in these specimens. Fig. 4. Open in new tabDownload slide Scanning electron micrographs of osmium-macerated human leucocytes. (a) Lymphocyte. A spherical nucleus (purple) is surrounded by a thin layer of cytoplasm (yellow). Green, mitochondria. The blood cell is perfectly surrounded by the agarose medium (filled star). (b) Neutrophil with lobulated nucleus (purple). Specific and azurophilic granules are observed throughout the cytoplasm. The boxed area is enlarged in (c). Filled star, agarose medium. (c) Closer view of a part of (b). The structures of the Golgi apparatus (blue) and mitochondria (green) are clearly observed in three dimensions. Purple, nucleus; red, specific and azurophilic granules; yellow, cytoplasm. Fig. 4. Open in new tabDownload slide Scanning electron micrographs of osmium-macerated human leucocytes. (a) Lymphocyte. A spherical nucleus (purple) is surrounded by a thin layer of cytoplasm (yellow). Green, mitochondria. The blood cell is perfectly surrounded by the agarose medium (filled star). (b) Neutrophil with lobulated nucleus (purple). Specific and azurophilic granules are observed throughout the cytoplasm. The boxed area is enlarged in (c). Filled star, agarose medium. (c) Closer view of a part of (b). The structures of the Golgi apparatus (blue) and mitochondria (green) are clearly observed in three dimensions. Purple, nucleus; red, specific and azurophilic granules; yellow, cytoplasm. Discussion The present study has introduced a useful method for observing three-dimensionally the internal structure of osmium-macerated cultured and free blood cells by SEM. The agarose embedment can be used for conventional LM and TEM. This method is also useful for analyzing SEM findings and comparing them with those obtained by LM and TEM. Osmium-maceration methods were originally introduced for direct SEM observation of cell organelles in tissue blocks, but it has been quite difficult to apply these methods to SEM studies of free cells. This is mainly because free cells must be supported or embedded in some material that should be resistant to prolonged osmium treatment. For this purpose, Fukudome and Tanaka [10] introduced a chitosan-embedding method and stated that commonly used embedding materials such as gelatin, agar and egg albumin are not of use because they dissolved during osmium maceration. In contrast, Isola et al. [11] have recently used low-melting-point agarose as an embedding material for C. albicans and have succeeded in observing the internal structure of this yeast by SEM after osmium maceration. In the present study, we clearly showed that low-melting-point agarose was a useful embedding medium for osmium maceration not only for microorganisms, but also for free cells. This is because agarose is not dissolved during osmium treatment. Agarose is also suitable for our study, because it can completely fill the space between the cells without penetrating them, even though they are not covered by a cell wall, as in C. albicans. Furthermore, when compared with a chitosan-embedding method, our agarose-embedding procedure is simple and rapid. Agarose is the principal polysaccharide component of agar, and is being used in gel electrophoresis. Yuan and Gulyas [13] have demonstrated that low-melting-point agarose can be used as an embedding medium for single cells (spermatozoa and oocytes) and dissociated cells (luteal and spleen cells). They embedded the agarose block in Epon and observed the fine structure of cells by TEM. In the present study, we also showed that this embedding did not cause any structural changes to the cells. This is probably partly because there was no heat damage in using low-melting-point agarose. Thus, embedding free cells in this material is a useful method for SEM; the 3D structure of free cells can be analyzed by SEM, the image of which was also compared with TEM images using the same fixation and embedding conditions. Another merit of using low-melting-point agarose is its convenience of handling during embedding. Conventional agarose or agar forms a gel below 37–40°C, whereas low-melting-point agarose is hardened below 28°C. Thus, the viscosity of low-melting-point agarose is sufficiently low for embedding, even at temperatures below 40°C. As a result, cells can be completely mixed in the medium at 35–40°C, even though the concentration of agarose is rather high (5%). Harvesting of cultured cells is important for SEM observation of their internal structures. In order to collect cultured cells, they must be detached from the culture plate by enzyme treatment. However, enzyme treatment has a risk of causing damage to the cell. Thus, we used temperature-responsive culture dishes in the present study. Cells cultured on these dishes can be detached by reducing the temperature to 20°C without any enzyme treatment and the dishes have been recently used in a variety of biological studies [14–16]. Temperature-responsive dishes were also useful for our studies because cultured cells can easily be collected for agarose embedding. Furthermore, Shimizu et al. [17] have recently introduced a supporting membrane (Cell Shifter; Cell Seed, Tokyo, Japan) for temperature-responsive dishes and have demonstrated that cultured cells (e.g. pancreatic islet cells) can be harvested as a tissue sheet. Our preliminarily experiment showed that combination of this method and our agarose-embedding method is useful for observing the internal structure of layered culture cells by both SEM and TEM, the detail of which will be described in a separate study. Conclusion The present study has introduced a useful method for observing 3D intracellular structures of cultured and free cells (i.e. HeLa and human blood cells) by SEM after osmium maceration. This method is simple and rapid when compared with the previous chitosan-embedding method and has the merit of analyzing the intracellular membranous organelles in cultured and/or free cells by SEM in relation to the findings made by LM and TEM of the same embedded samples. Thus, this method is expected to be widely used for the 3D analysis of intracellular structures in cells such as leukocytes, spermatozoa and various kinds of cultured cells, as well as yeast and other microorganisms. Funding This work was supported by a Grant for Promotion of Niigata University Research Projects (234058). References 1 Tanaka K , Naguro T . High resolution scanning electron microscopy of cell organelles by a new specimen preparation method , Biomed. Res. , 1981 , vol. 2 Suppl. (pg. 63 - 70 ) OpenURL Placeholder Text WorldCat 2 Tanaka K , Mitsushima A . A preparation method for observing intracellular structures by scanning electron microscopy , J. Microsc. , 1984 , vol. 133 (pg. 213 - 222 ) 10.1111/j.1365-2818.1984.tb00487.x Google Scholar Crossref Search ADS PubMed WorldCat Crossref 3 Hanaki M , Tanaka K , Kashima Y . Scanning electron microscopic study on mitochondrial cristae in the rat adrenal cortex , J. Electron Microsc. , 1985 , vol. 34 (pg. 373 - 380 ) OpenURL Placeholder Text WorldCat 4 Tanaka K , Mitsushima A , Fukudome H , Kashima Y . Three-dimensional architecture of the Golgi complex observed by high resolution scanning electron microscopy , J. Submicrosc. Cytol. , 1986 , vol. 18 (pg. 1 - 9 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 5 Lea P , Hollenberg M J . Mitochondrial structure revealed by high-resolution scanning electron microscopy , Am. J. Anat. , 1989 , vol. 184 (pg. 245 - 257 ) 10.1002/aja.1001840308 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 6 Tanaka K , Fukudome H . Three-dimensional organization of the Golgi complex observed by scanning electron microscopy , J. Electron Microsc. Tech. , 1991 , vol. 17 (pg. 15 - 23 ) 10.1002/jemt.1060170104 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 7 Isola R , Solinas P , Loy F , Mariotti S , Riva A . 3-D structure of mitochondrial cristae in rat adrenal cortex varies after acute stimulation with ACTH and CRH , Mitochondrion , 2010 , vol. 10 (pg. 472 - 478 ) 10.1016/j.mito.2010.05.007 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 8 Riva A , Tandler B , Ushiki T , Usai P , Isola R , Conti G , Loy F , Hoppel CL . Mitochondria of human Leydig cells as seen by high resolution scanning electron microscopy , Arch. Histol. Cytol. , 2010 , vol. 73 (pg. 37 - 44 ) 10.1679/aohc.73.37 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 9 Koga D , Ushiki T . Three-dimensional ultrastructure of the Golgi apparatus in different cells: high-resolution scanning electron microscopy of osmium-macerated tissues , Arch. Histol. Cytol. , 2006 , vol. 69 (pg. 357 - 374 ) 10.1679/aohc.69.357 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 10 Fukudome H , Tanaka K . A method for observing intracellular structures of free cells by scanning electron microscopy , J. Microsc. , 1986 , vol. 141 (pg. 171 - 178 ) 10.1111/j.1365-2818.1986.tb02713.x Google Scholar Crossref Search ADS PubMed WorldCat Crossref 11 Isola M , Isola R , Lantini M S , Riva A . The three-dimensional morphology of Candida albicans as seen by high-resolution scanning electron microscopy , J. Microbiol. , 2009 , vol. 47 (pg. 260 - 264 ) 10.1007/s12275-008-0212-1 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 12 Murakami T . A metal impregnation method of biological specimens for scanning electron microscopy , Arch. Histol. Jap. , 1973 , vol. 35 (pg. 323 - 326 ) 10.1679/aohc1950.35.323 Google Scholar Crossref Search ADS WorldCat Crossref 13 Yuan L , Gulyas B J . An improved method for processing single cells for electron microscopy utilizing agarose , Anat. Rec. , 1981 , vol. 201 (pg. 273 - 281 ) 10.1002/ar.1092010207 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 14 Nishida K , Yamato M , Hayashida Y , Watanabe K , Maeda N , Watanabe H , Yamamoto K , Nagai S , Kikuchi A , Tano Y , Okano T . Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface , Transplantation , 2004 , vol. 77 (pg. 379 - 385 ) 10.1097/01.TP.0000110320.45678.30 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 15 Ide T , Nishida K , Yamato M , Sumide T , Utsumi M , Nozaki T , Kikuchi A , Okano T , Tano Y . Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes , Biomaterials , 2006 , vol. 27 (pg. 607 - 614 ) 10.1016/j.biomaterials.2005.06.005 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 16 Sekiya S , Shimizu T , Yamato M , Kikuchi A , Okano T . Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential , Biochem Biophys Res Commun , 2006 , vol. 341 (pg. 573 - 582 ) 10.1016/j.bbrc.2005.12.217 Google Scholar Crossref Search ADS PubMed WorldCat Crossref 17 Shimizu H , Ohashi K , Utoh R , Ise K , Gotoh M , Yamato M , Okano T . Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus , Biomaterials , 2009 , vol. 30 (pg. 5943 - 5949 ) 10.1016/j.biomaterials.2009.07.042 Google Scholar Crossref Search ADS PubMed WorldCat Crossref © The Author 2012. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved. 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TI - A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfr098
DA - 2012-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-useful-method-for-observing-intracellular-structures-of-free-and-NVHLTEkYOL
SP - 105
EP - 111
VL - 61
IS - 2
DP - DeepDyve
ER -