Culture of cells in beem capsules: A new technique for electron microscopic study of monolayer cultures

Summary A method is described for the monolayer cultivation of primary cell suspensions and established cell lines directly in carbon-coated BEEM capsules. BEEM capsules are routinely employed by electron microscopists in tissue embedding procedures; growing monolayer cultures directly on the lids of inverted BEEM capsules presents the obvious advantage of maintaining cell to cell to substratum contacts with a minimum of stress and damage in the preparative steps for electron microscopy. This work was supported by grant AM 17631 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, grant CA 11339 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

An Improved Method of Making Tissue Carriers for TEM and SEM Fixation

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

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Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

A Device for Flat-Face, Epoxy Resin Embedding of Tissues and Coverslip Cultures

An easily constructed device permits the flat-face embedding of four specimens in epoxy resins. Either pieces of tissue or cells grown on cover slips can be used. After polymerization, the flat-surfaced capsules may be examined under high magnification for selection of areas to be sectioned. Specimens difficult to orient can be cut out and re-embedded in the proper position. The use of thick-walled BEEM capsules and the clamping action afforded by four screws prevent leakage of the resin.     

A PLastic MIcrostrainer for HAndling MAmmalian BLastocysts

Friend (1963) has described a microstrainer for handling microscopic marine ova which permits direct transfer of batches of ova through various histological solutions without centrifugation or decanting. This strainer consists of a length of glass tubing with an electron microscope grid cemented over one end. In our laboratory, several modifications of this device have been made for use in handling mammalian ova and blastocysts. The most satisfactory model, which can be made easily, was contructed from BEEM plastic capsules (Better Equipment for Electron Microscopy, Inc., P.O. Box 132 Jerome Avenue Station, Bronx, New York 10468) and circular electron microscope grids of suitable mesh size.     

Direct Epoxy Embedding for Vertical Sectioning of Cells Grown as a Monolayer on Millipore Filter

Cells cultured as a monolayer on MF-Millpore GSWP. 0.22 μ pore size, filter were fixed, dehydrated, and examined by phase-contrast microscopy with the filter immersed in a 1:1 mixture of xylene and the embedding medium. The membrane was cut into 2 × 20 mm strips, and each strip which was selected for desired cells was embedded vertically in a BEEM capsule. Thus direct embedding which allowed edgewise sectioning of cells was obtained without removing them from the culturing support.     

Isolation and Embedding of Single Nerve Cells for Electron Microscopy

Individual neurons have been isolated by freehand dissection under binocular, 40-100 power magnification from the lateral vestibular nucleus of fresh rabbit brain. The cells were fixed with glutaraldehyde and OsO₄ and dehydrated in 33%, 50%, 60% (v/v) and pure Durcupan A prior to embedding in Araldite 502. Blocks were cast in lids of BEEM capsules. The locating of the individually embedded cells and trimming of the blocks were facilitated by means of a holder that permitted visualization in both diffuse transmitted and incident light.     

Gold labeling of cell monolayers grown on dextran beads

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.     

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

Direct embedding in epoxy resin of cells attached to cellophane

Macrophages were collected from mice or rats after subcutaneous implantation of cellophane or grown in Leighton tubes in which the flying coverslip was replaced by a strip of cellophane. The cellophane and attached cells were prepared for electron microscopy, embedded directly in epoxy resin and then sectioned. The cutting qualities of cellophane are adquate for ultrathin sectioning and permit the ultrastructural examination of the attached macrophage monolayer. The technique can be used for other cell types.     

In situ embedding method of cells grown in beem capsules for immunoelectron microscopic studies of oncornaviruses.

A technique of in situ embedding of cells grown in BEEM capsules has been devised for immunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Dm strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues.     

Growth factor production from fibrin-encapsulated human keratinocytes

Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-β1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-β1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-β1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-β1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.     

A Squeeze-Out Method,Using Pliers,for Removing Hardened Blocks from Polyethylene Capsules

The original BEEM capsule #1000 (Better Equipment for Electron Microscopy, P.O. Box 132, Jerome Station, Bronx, NY 10469) is now widely used as a casting mold in plastic embedding. This polyethylene capsule has proven popular with many electron microscopists because it provides a preshaped truncated pyramid casting that requires minimal trimming prior to sectioning. A disadvantage of the plastic capsule is that it must be either laboriously slit or partially cut away to allow removal of the hardened block; or, one may use a special, rather costly press designed for such removal.     

Growth of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. immobilized in chitosan with different times of contact with NaOH

The thickness of the walls of the capsules of chitosan-immobilized Synechococcus cultures was dependent on the time of contact with NaOH and was directly related to culture growth. After an initial lag phase, probably caused by cell damage, the capsules obtained after 80 s in a 0.1 N NaOH solution showed better growth than that of free cell cultures (6.9 and 5.2 divisions in 10 days, respectively).     

Strategy for the Use of Affinity Grids to Prepare Non-His-Tagged Macromolecular Complexes for Single-Particle Electron Microscopy

Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized nickel-nitrilotriacetic acid lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here, we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II, still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-Å-resolution density map by single-particle cryo-EM.     

Rapid polymerisation with microwave irradiation for transmission electron microscopy

Successful results of microwave polymerisation of different epoxy formulations have been reported in the literature. The present study was intended to shorten the time needed for polymerisation of epoxy resin by the use of a microwave technique. A standard double fixation and tissue processing was applied to samples of rat kidney tissue. Tissue samples from the control group were polymerised in a conventional oven at 60 degrees C for 48 h, while tissue from the experimental group was irradiated in a microwave oven, initially at 900 W for 10 min and then at 360 W for another 100 min. During this irradiation, the sealed BEEM capsules were submerged in a water bath, so that the temperature rise was uniform and constant. This resulted in a homogeneous and rapid polymerisation. The cutting properties of the blocks in both groups were similar and no noticeable difference in the quality of the sections was evident when evaluated with TEM. The results showed that the use of a microwave oven reduced the time needed for the polymerisation of Epon blocks without any loss in quality.     

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133⁺/CD44⁺ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133⁺/CD44⁺ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.     

Methyl Green-Pyronin for Staining Autoradiographs of Hydroxyethyl Methacrylate-Embedded Lymphoid Tissue

Cells in the spleen in DNA-synthesis were labelled with tritiated thymidine. Tissue was fixed for 12 hr in 10% neutral formalin, washed for 4 hr in tap water and dehydrated through 70% and absolute ethanol. The tissue blocks were infiltrated overnight with a mixture consisting of glycol methacrylate, 80 ml; polyethylene glycol 400, 12 ml; and benzoyl peroxide, 0.27 gm. Specimens were cast in BEEM capsules with the final embedding medium consisting of 42 parts of the infiltration medium and 1 part of an acceleration mixture. This mixture consisted of N,N-dimethylaniline, 1 part and polyethylene glycol 400, 15 parts. The blocks hardened in 30 min and were sectioned with an ultramicrotome fitted with glass knives. Sections were coated with Ilford K5 liquid emulsion and exposed for 2 wk. Methyl green-pyronin staining of autoradiographs was carried out at pH 4.1 in acetate buffer containing 0.5% methyl green (Allied Chemicals) and 0.2% pyronin GS (Chroma). Staining was for 30-60 min, after which sections were washed for 1 min in water, blotted, allowed to dry, and mounted in Canada balsm. The procedure resulted in good quality autoradiographs in which the degree of basophilia of labelled cells could be assessed.     

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.