A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.     

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.     

Mouse hepatitis virus immunofluorescence in formalin- or Bouin's-fixed tissues using trypsin digestion

Mouse hepatitis viral antigens were demonstrated by immunofluorescence in formalin- and Bouin's-fixed tissues processed routinely for histopathology followed by partial digestion with trypsin. Staining was superior in tissues fixed in formalin and was not diminished in tissue sections from paraffin blocks stored at room temperature as long as 2 years. The relative ease of this procedure and the commercial availability of reagents makes this a useful technique for the definitive diagnosis of mouse hepatitis virus infection.     

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathologic study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tissue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.     

Establishment of primary cell cultures from fish calcified tissues

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.     

Dissociation of single cells from lung or kidney tissue with elastase

Summary Experiments were conducted to determine the capacity of various enzyme preparations to dissociate single cells from guinea pig lung tissue. The number of cells separated from tissue progressively increased as the concentration of crude trypsin was increased from 25 to 250 mg per 100 ml. This action could be inhibited by soy bean trypsin inhibitor. Elastase, but not ethylenediaminetetraacetate (disodium salt), crystalline trypsin, nor chymotrypsin, dissociated cells from lung tissues. Crude trypsin (Trypsin 1∶300) was found to contain 3.0 Sachar units of elastase per mg. Elastase was also inhibited by soy bean trypsin inhibitor. Only some collagenase preparations dissociated cells from lung tissue. Impure bacterial proteases dissociated lung cells. Our data suggest that the term “trypsinization” to denote dissociation of cells from tissue with crude preparations of trypsin is misleading and should be discontinued. Partially supported bv Armour-Baldwin Laboratories and the National Institute of Health, Grant, AM 12919.     

Efficient reactivation of latent herpes simplex virus from mouse central nervous system tissues

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.     

Spontaneous and lectin-induced redistribution of cell surface receptors on embryonic chick neural retina cells.

The mobility of plant lectin receptors in the plane of the membrane is examined for cells prepared from embryonic chick neural retinas by a variety of procedures. Cells liberated from the intact tissue by trypsin treatment followed by mechanical dissociation are able to redistribute their receptors into 'caps' both spontaneously and in the presence of a multivalent lectin. These cells, dispersed by trypsinization, upon repair in culture for a suitable period of time lose their ability to redistribute lectin receptors. Cells dispersed by mechanical means without prior trypsin treatment are unable to undergo 'cap' formation. In addition, cells within intact tissues are also unable to redistribute their lectin receptors into 'caps.' Based on these observations we propose that within solid tissues which have assumed their characteristic architecture, cell surfaces are immobilized, and that this phenomenon may be a critical parameter in determining the potential of a cell to undergo morphogenetic rearrangements.     

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique.     

Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.     

Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR

Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA (rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C(T) values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.     

Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A

Summary The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue.The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.     

Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp.

Effects of the enzymes trypsin, papain, bromelains and ficin on bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17 degrees, 20 degrees and 37 degrees C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle's MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca2+ and Mg2+.     

Studies on autoimmune encephalomyelitis in the guinea pig--III. A comparative study of two autoantigens of central nervous system myelin.

Abstract— A new CNS myelin autoantigen(s) (referred to as M2), different from the encephalitogenic basic protein (BP), can be detected with guinea-pig demyelinating and complement fixing (CF) sera raised against guinea pig CNS tissue or myelin (Lebar et al., 1976). M2 and BP were present in mouse, rat, rabbit, bovine and human CNS tissues when tested with guinea-pig homologous specific antisera; they were not present in non-CNS tissues. Both autoantigens were also detected in newborn guinea-pig myelin and myelin-like fractions. The CF activity of myelin with demyelinating (anti-M2) sera was not altered by trypsin; however, absorption experiments showed that M2 was partly trypsin sensitive. Both antibodies against the trypsin sensitive and the trypsin resistant determinants of M2 were demyelinating. Both determinants of M2 were preselit in mouse, rat, rabbit, bovine‘and human CNS tissues and in guinea-pig newborn myelin. CF BP activity of myelin was partially or even totally abolished by trypsin, but the persistent encephali-togenicity of trypsin-treated myelin could be attributed to non-CF encephalitogenic peptides from BP. In accordance with recent work our results tend to support an inner localization of BP in myelin; M2, on the other hand, would be a surface antigen(s).     

Multiple regeneration from axolotl limb stumps bearing cross-transplanted minced muscle regenerates.

Flexor and extensor muscles in the upper arms of axolotls were minced and cross-transplanted. The limbs were amputated 5 and 30 days after mincing. In each experiment a high percentage of the regenerates consisted of multiple limbs. This demonstrates that the morphogenetic information which produces multiple regenerates after the cross-transplantation of limb stump muscle is stable enough to be expressed after both mincing and the subsequent tissue regeneration of the muscle.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎（RA）动物模型;从炎性关节滑膜中分离成纤维样滑膜细胞（FLS）。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎;剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似;成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

组织单腺体内单细胞的基因变异分析方法

从单细胞尺度进行细胞异质性的分析是深度理解细胞群体关系的关键。组织中的单细胞由于细胞类型不同,尺寸往往相差很大,但是目前常用的基于微孔板和Fluidigm公司的微流控的单细胞组学研究方法,需要入口的单细胞大小相近。本研究以胃组织为例,建立了一种组织单细胞的基因变异分析方法,实现了尺寸差异较大的单细胞的基因变异分析。在该方法中,先将胃组织裂解获得单个腺体,再将单个腺体酶解得到不同大小的腺体内单细胞,然后把这些单细胞铺在聚乙烯萘膜载玻片上,进行激光显微切割分选、全基因组放大,最后测其微卫星的长度。利用该方法,成功在肠上皮化生腺体内部检测到微卫星长度的变化,并灵活地对尺寸差异大的组织细胞以及肠化生腺体细胞进行了精细分析。此外,这种单细胞分析方法还可以对带有不同标记的细胞进行低通量和高通量的基因组分析,为单细胞尺度上的组织异质性研究提供了一种高度灵活的分析方法。     

Proteolysis and lectin histochemistry

Summary Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used.Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified -chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffinembedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied.These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.