Agarose Embedding: A New Method for the Ultrastructural Examination of the In-Situ Morphology of Cell Cultures

A new technique for the ultrastructural examination of the in-situ morphology of cell cultures is described. By embedding the cell cultures in situ in agarose before embedding them in epoxy resin, numerous agarose blocks with the cells on top of the blocks can be obtained and used for different types of investigation, including electron microscopy. Use of this technique enables many of the problems encountered with earlier methods to be avoided.     

Agarose mold embedding of cultured cells for tissue microarrays.

There are several indications for the placement of samples of cultured cells in tissue microarrays (TMAs). To optimize this technique, three embedding procedures were compared: embedding of fixed cells pelleted by centrifugation, embedding of cells dispersed in an agarose matrix, and embedding of pelleted cells packed into the center of hollow agarose molds. TMAs were made from these preparations. The number of cells per tissue spot and the number of histologic sections that could be obtained from the preparations were determined. The agarose matrix and agarose mold techniques resulted in the longest core samples, while the cell pellet and agarose mold methods resulted in the greatest cell density. Thus, the use of cylindrical agarose molds optimizes both the number of cells present on a histologic section of a TMA, and the number of histologic sections that can be obtained from a TMA. This technique results in a paraffin-embedded cell preparation that yields a cell density of approximately 1000 cells per 0.6-mm diameter circular histologic section, and that produces uniform core samples the full thickness of the donor block. Histologic sections of TMAs prepared in this manner were validated in immunohistochemical and in situ hybridization assays.     

一种优化的琼脂糖包埋细胞微阵列的制备方法

在原有琼脂糖包埋细胞微阵列制备技术基础上,建立一种高可靠性和稳定性的细胞微阵列制备方法。方法是收集培养细胞,采用优化的低熔点琼脂糖包埋技术包埋目的细胞,再对细胞包埋块进行脱水和石蜡包埋,最后将细胞石蜡包埋块用组织微阵列仪制备成细胞微阵列。结果显示细胞微阵列切片HE染色细胞结构清晰,免疫组化实验后观察阳性信号着色明显,定位准确,无背景干扰,细胞微阵列在4℃存放3个月后进行免疫组织化学实验,信号无明显衰减。本方法制备的细胞微阵列具有较好的稳定性和可靠性,且细胞形态和抗原保存良好,可应用于生物科技和医学研究领域各种实验研究。     

Value of the innovated technique agarose cell block in improving the sensitivity of urine cytology in cases of bladder carcinoma

Proper handling and processing of urine sample can greatly improve diagnostic sensitivity. This work investigates the value of agarose cell block technique in processing urine samples simultaneously for light and electron microscopic examination, with the prospect to enhance the quality of diagnosis. The material of this study consisted of 45 voided urine samples, processed for the performance of Papanicolaou-stained urine smears, agarose cell blocks paraffin sections stained with hematoxylin & eosin, and electron microscopy-contrasted ultrathin sections. The studied technique increases the sensitivity of urine cytology and opens a new prospect for cytomorphological study.     

Value of the Innovated Technique Agarose Cell Block in Improving the Sensitivity of Urine Cytology in Cases of Bladder Carcinoma

Proper handling and processing of urine sample can greatly improve diagnostic sensitivity. This work investigates the value of agarose cell block technique in processing urine samples simultaneously for light and electron microscopic examination, with the prospect to enhance the quality of diagnosis. The material of this study consisted of 45 voided urine samples, processed for the performance of Papanicolaou-stained urine smears, agarose cell blocks paraffin sections stained with hematoxylin & eosin, and electron microscopy-contrasted ultrathin sections. The studied technique increases the sensitivity of urine cytology and opens a new prospect for cytomorphological study.     

Agarose cell block: innovated technique for the processing of urine cytology for electron microscopy examination

Easy manipulation and preservation of cells in suspension through the different steps of sample processing for electron microscopy examination is essential for proper diagnosis. The author used agarose gel as an embedding media for processing cells in suspension for electron microscopic examination. The AgarCyto cell block procedure of Kerstens et al. (J Histochem Cytochem. 2000; 48: 709—718) was used to begin electron microscopic processing of exfoliated urothelial cells in voided urine or cells in suspension. Processing of agarose cell block simultaneously for light and electron microscopic examination represents a great advantage offered by this innovated technique.     

塑料包埋技术在骨组织研究中的应用

目的研究塑料包埋技术制作的不脱钙硬组织切片,确定制作的关键步骤及用于骨组织研究的优点。方法选取狗胫骨节段或带有金属种植体的骨材料塑料包埋,LeicaSP1600切片机（德国）切片,用苦味酸品红染色观察,并与常规石蜡包埋相比较。结果不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、多孔材料及种植体周围骨组织的整合程度。与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用规范塑料包埋技术制作不脱钙硬组织切片,更有利于行骨形态计量分析以及材料与骨整合的研究。     

An improved method for processing single cells for electron microscopy utilizing agarose

An improved method is presented for processing single cells for electron microscopy. Agarose, which has a low (30°C) gelling temperature, was used as an initial embedding medium for single cells (spermatozoa and oocytes) and dissociated cell preparations (luteal cells and spleen cells). Dispersed cells of corpus luteum, spleen, and epididymal spermatozoa were placed in 1.5% agarose after aldehyde fixation. These fixed cells, embedded in agarose, were packed into a dense pellet by centrifugation, postfixed, then embedded in Epon. Mammalian eggs were not centrifuged; instead, they were embedded in agarose discs. Cells embedded in agarose were cooled below 30°C to allow for gelling, then processed for electron microscopy. Because agarose has a low gelling temperature, some heat-labile substances were preserved, as demonstrated by retention of peroxidase activity using the DAB histochemical method. The agarose embedding procedure is both rapid and facile, and has proven to be of value in the handling of fragile single cells for electron microscopic studies.     

Utility of the thromboplastin‐plasma cell‐block technique for fine‐needle aspiration and serous effusions

(I) To assess the feasibility of thromboplastin‐plasma (TP) method for cell block, (II) to concentrate the minimal cellular material from effusions and needle‐rinses by block preparation and improve visual details, (III) to compare conventional cytological smears with cell blocks for final assessment, and (IV) to assess utility of immunocytochemistry (ICC) for diagnostic accuracy. Seventy cell blocks were prepared by TP technique using surplus fluid from 38 serous effusions, and for 32 ultrasonography‐guided fine‐needle aspiration cytology (FNAC) material, rinses of syringes and needles were collected in normal saline after conventional cytological smears. Then, cell blocks were compared with conventional smears for adequacy, morphologic preservation, and ICC. Absolute concordance seen in 66 cases (94%) between the smears and cell blocks. Advantages with the blocks were cellular concentration in a limited field and better cellular preservation with architectural pattern. Quality of ICC was comparable to that of standard controls. Diagnostic discrepancy was seen in two cases where cell blocks were positive but smears were negative. Two cell blocks were nonrepresentative. Cell block serves as a useful adjunct to traditional cytological smears. TP method is simple, cost effective, and reproducible. It is easy when compared with agar‐embedding technique. Ancillary techniques like ICC can be performed successfully. Diagn. Cytopathol. 2009. © 2008 Wiley‐Liss, Inc.     

Further characterization of herpes virus persistence.

Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.     

Detection of HIV envelope specific antibodies by immunoelectron microscopy and correlation with antibody titer and virus neutralizing activity

Human antisera positive for HIV were evaluated on HTLV-IIIB producing cells by two different immunoelectron microscopic (IEM) techniques. In preembedding immunoferritin IEM a heavy label was observed with early budding HIV. Under the same conditions cell released 'mature' particles were almost negative, which could be explained by the direct observation that most of the surface glycoprotein knobs are lost spontaneously during virus maturation. Using freshly infected cultures after agarose embedding, immunogold labelling of ultrathin cryosections allowed us to detect and differentiate internal core as well as virus envelope antigens. A good qualitative correlation between neutralization titers and IEM labelling intensity was observed. This type of immunocryoultramicrotomy appears to be useful for the detection of antigens in and on the virion. It might turn out valuable for the characterization of the env gp120 epitopes of HIV.     

塑料包埋结合四环素荧光标记制作不脱钙骨组织切片的实验研究

目的探讨塑料包埋技术结合四环素荧光标记制作不脱钙骨组织切片的关键步骤及应用。方法选取四环素荧光标记的兔股骨进行塑料包埋,Leica SP 1600切片机切片,荧光显微镜观察后用苦味酸品红染色,并与常规石蜡包埋切片相比较。结果四环素荧光标记不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、植入体及周围骨组织的整合程度,与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用塑料包埋技术制作四环素荧光标记不脱钙骨组织切片,有利于行骨形态计量分析以及植入体与骨整合的研究。     

Cell culture block array for immunocytochemical study of protein expression in cultured cells.

Immunocytochemical staining of cultured cells using specific antibodies is a powerful technique to study the expression and subcellular localization of proteins. However, this technique is associated with sample-to-sample variations because samples are handled individually and manually. Cell permeation is needed when intracytoplasmic or nuclear proteins are studied. Storage of cultured cells is difficult, and experiments must be repeated if additional studies are desired later, which introduces more variations. We developed a cell culture block array technique that converts cultured cells into a permanently fixed form identical to tissue sections prepared for pathologic examination. Cells from different cultures can be embedded in a single block. Many identical sections, each containing cells from multiple cultures, may be stained with different antibodies using an automated stainer. As a result, sample-to-sample variation is eliminated. Because cells in these blocks are sectioned by knives, all cellular proteins come into direct contact with antibodies, and cell permeation is not needed. Such blocks can be conveniently stored for years without loss of antigens, providing a constant source for future studies. We demonstrated the utility of this technique by studying the proliferation and neuroendocrine differentiation of prostate cancer-derived LNCaP cells cultured in vitro.     

Immunocytochemical detection of Candida albicans in formalin fixed, paraffin embedded material

AIM: To assess the ability of the commercially available monoclonal antibody 1B12 (BioGenex, San Ramon, USA) to identify C albicans in formalin fixed, paraffin wax embedded material (FFPE). METHODS: Broth cultures of 20 strains of seven Candida species were resuspended in 4% agarose blocks, fixed in formalin for 24 hours, and embedded in paraffin wax. In addition, 16 blocks of FFPE tissue known to contain periodic acid-Schiff positive fungal hyphae were examined. Antigen retrieval involved microwave treatment of specimens in citrate buffer (0.01 M; pH 6.5) before addition of 1B12 antibody for 24 hours. Bound antibody was subsequently detected using a biotinylated link antibody and a peroxidase conjugated streptavidin. RESULTS: Only C albicans strains were 1B12 positive in the agarose blocks. All FFPE tissue blocks were found to contain 1B12 positive hyphal structures, indicating the presence of C albicans. CONCLUSIONS: The ability to identify candida organisms penetrating the lesional tissue in cases of chronic hyperplastic candidosis will help to clarify the role of individual Candida spp in this important form of oral candidosis.     

Progressive changes in T₁, T₂ and left-ventricular histo-architecture in the fixed and embedded rat heart

Chemical tissue fixation, followed by embedding in either agarose or Fomblin, is common practice in time-intensive MRI studies of ex vivo biological samples, and is required to prevent tissue autolysis and sample motion. However, the combined effect of fixation and sample embedding may alter tissue structure and MRI properties. We investigated the progressive changes in T(1) and T(2) relaxation times, and the arrangement of locally prevailing cardiomyocyte orientation determined using diffusion tensor imaging, in embedded ex vivo rat hearts fixed using Karnovsky's solution (glutaraldehyde-formaldehyde mix). Three embedding media were investigated: (i) standard agarose (n = 3 hearts); (ii) Fomblin (n = 4 hearts); and (iii) iso-osmotic agarose (n = 3 hearts); in the latter, the osmolarity of the fixative and embedding medium was adjusted to 300 mOsm to match more closely that of native tissue. The T(1) relaxation time in the myocardium showed a pronounced decrease over a 48-h period following embedding in Fomblin (-11.3 ± 6.2%; mean ± standard deviation), but was stable in standard agarose- and iso-osmotic agarose-embedded hearts. The mean myocardial T(2) relaxation time increased in all embedded hearts: by 35.1 ± 14.7% with standard agarose embedding, 13.1 ± 5.6% with Fomblin and 13.3 ± 1.4% with iso-osmotic agarose. Deviation in the orientation of the primary eigenvector of the diffusion tensor occurred in all hearts (mean angular changes of 6.6°, 3.2° and 1.9° per voxel after 48 h in agarose-, Fomblin- and iso-osmotic agarose-embedded hearts, respectively), indicative of progressive structural changes in myocardial histo-architecture, in spite of previous exposure to fast-acting tissue fixation. Our results suggest that progressive structural changes occur in chemically fixed myocardium, and that the extent of these changes is modulated by the embedding medium, and by osmotic gradients between the fixative in the tissue and the surrounding medium.     

Hydrogel effects on bone marrow stromal cell response to chondrogenic growth factors

The aim of this study was to investigate the effects of alginate and agarose on the response of bone marrow stromal cells (BMSCs) to chondrogenic stimuli. Rat BMSCs were expanded in monolayer culture with or without FGF-2 supplementation. Cells were then seeded in 2% alginate and agarose gels and cultured in media with or without TGF-beta1 or dexamethasone (Dex). Sulfated glycosaminoglycans (sGAGs), collagen type II, and aggrecan were expressed in all groups that received TGF-beta1 treatment during hydrogel culture. Expansion of rat BMSCs in the presence of FGF-2 increased production of sGAG in TGF-beta1-treated groups over those cultures that were treated with TGF-beta1 alone in alginate cultures. However, in agarose, cells exposed to FGF-2 during expansion produced less sGAG within TGF-beta1-supplemented groups over those cultures treated with TGF-beta1 alone. Dex was required for optimal matrix synthesis in both hydrogels, but was found to decrease cell viability in agarose constructs. These results indicate that the response of BMSCs to a chondrogenic growth factor regimen is scaffold dependent.     

Enhancement of varicella-zoster virus plaquing efficiency with an agarose overlay medium

The infectivity titers of varicella-zoster virus (VZV) are routinely estimated by plaque production in cell culture. In this report, we show that plaque counts for VZV (strain Oka/Merck), in MRC-5 cell cultures, are significantly enhanced (54% average enhancement) by the use of an agarose overlay medium, as compared to a fluid overlay medium. Evidence also is presented that less variability (P less than 0.05) in plaque counts occurs with the use of an agarose overlay medium.     

Formaldehyde–hardened albumin as a non-penetrating embedding matrix for frozen and vibratome sectioning

In this paper, we describe a protocol for a non-penetrating embedding matrix that can be used for frozen or vibratome sectioning of various formaldehyde-fixed tissue specimens. In our experiments, we wanted to prepare thin frozen sections from miniature specimens for fluorescent staining. As we could not achieve satisfactory results with any of the previously published methods, we have tried to modify the existing protocols, and systematically evaluated the effect of these modifications on the properties of the embedding matrix. The resulting protocol is simple, the matrix gets firmly attached to the tissues, does not cause autofluorescence and enables preparing extremely thin frozen sections. The matrix can be used for 1, embedding miniature specimens from problematic tissues to enable cutting very thin frozen sections, 2, grouping multiple specimens into one large block for simultaneous processing, and 3, dispersing single cells and preparing cell blocks for frozen sectioning.     

Comparison of scaffolds and culture conditions for tissue engineering of the knee meniscus

The menisci of the knee are semilunar fibrocartilaginous structures critical in load bearing, shock absorption, stability, and lubrication. In this study, two commonly used biomaterials, a hydrogel (agarose) and a nonwoven mesh polymer [poly(glycolic acid); PGA], were compared for suitability as scaffold materials for tissue engineering the knee meniscus. In addition, a rotating wall bioreactor culture of both scaffold materials was compared with static cultures. Constructs were cultured for up to 7 weeks in static and rotating wall bioreactor culture. Cell numbers were 22 times higher in PGA than agarose after 7 weeks in culture. Static PGA scaffolds had more than twice the amount of sulfated glycosaminoglycans and three times the amount of collagen compared to static agarose constructs at week 7. The rotating wall bioreactor was not found with increase matrix production or cell proliferation significantly over static cultures.