Fibroblasts, tissue culture

For positive controls, it is extremely important to have a tissue culture cell line with an abundant amount of target nucleic acid. Varieties of tissue culture cell lines are available from American Tissue Culture Center, Rockville, MD. Rapidly growing tissue culture cell lines transfected with the target nucleic acid could be successfully used for positive controls. For example, formalin-fixed paraffin-embedded foreskin fibroblast tissue culture cell-line FS4 can be used as a positive control for human lysyl oxidase mRNA detection and c-W-ras transformed RS-485 cell line for normal ras message detection. Thin sections (4-5 im) of paraffin-embedded positive control cell lines could be placed simultaneously near the side of the experimental human or animal tissue. Similarly, one can also use freshly cut animal or human tissue (with the target sequences) preserved, sectioned, and mounted under standard laboratory conditions. For negative controls, choose a cell line (or a tissue) that completely lacks the target nucleic acid see Note 12). 

The synthetic tridecamer, which is complementary to 13 bases of the 3 - and 5 -reiterated terminal sequences of Rous sarcoma virus 35S RNA," was added to chick embryo fibroblast tissue cultures infected with Rous sarcoma virus. Inhibition of virus production resulted. This is because the synthetic oligomer hybridizes with the terminal reiterated sequences at the 3 -and 3 -ends of the 35S RNA and interferes with one or more steps involved in viral production and cell transformation. 

Fibroblast locomotion has been studied in tissue culture systems and is unexpectedly complex. Initially when fibroblasts are plated, they are rounded and... 

Abercrombie, M Heaysman, J.E.M. (1954). Social behavior of cells in tissue culture. II. Monolayering of fibroblasts. Exp. Cell Res. 6. 293-306. 

There has been an increase in the use of cadaveric heart valves for patients with valvular defects. The valves are best stored by freezing but some success has been achieved by simple cold storage in an antibiotic medium made up of ingredients common to most tissue culture solutions. At a storage temperature of 4 °C there is a continual loss of viability of fibroblasts so that by three weeks there are practically no viable cells and the valves cannot be used. 

Initially, the cytotoxicity against chick embryo fibroblasts of BPA, tyrosine, tyrosine dipeptide, and the dipeptide derivatives used in the synthesis of the polymers shown in Fig. 7 were evaluated in a comparative experiment (43). The surface of standard tissue culture wells was coated with 5 mg of each test substance. Then the adhesion and proliferation of the fibroblasts was followed over a 7-day period. Among all test substances, BPA was clearly the most cytotoxic material. Monomeric tyrosine derivatives containing the ben-zyloxycarbonyl group were also cytotoxic, while tyrosine itself, tyrosine dipeptide, and most of the protected dipeptide derivatives did not noticeably interfere with cell growth and adhesion and were therefore classified on a preliminary basis as possibly "nontoxic."... 

White blood cells, red blood cells and cultured fibroblasts are commonly used to measure enzyme activities, especially for the diagnosis of inherited enzyme abnormalities. Leukocytes may be collected by sedimentation in viscous media such as Fycol. The collection of red cells presents no problem following centrifugation of anticoagulated blood. The assay of enzymes and fibroblasts requires appropriate tissue culture facilities and extensive experience in dealing with cultured human cells. 

Among the many phosphorus compounds selected by Mitchell and his collaborators may be mentioned tetrasodium 2-methyl l 4-naphthohydroquinone diphosphate (VTH). A concentration of 4 x 10 6 M of (VIII) produced a 50 per cent mitotic inhibition using chick fibroblasts in tissue culture. It is thought that the inhibition of the entry of cells into mitosis depends on the blockage of cellular synthetic processes involving phosphoryla-... 

Fetner has also demonstrated chromatid breaks in a human tissue-culture cell line exposed to ozone at 8 ppm for 5 min. Other tissue-culture studies include that of Sachsenmaier et who noted tetraploidy and other chromosomal abnormalities in embryonic chick fibroblasts exposed to ozone and a decrease in transplantability of mouse ascites tumor cells. In addition, Pace et demonstrated an interference by ozone with mitotic activity in two tissue-culture cell lines. More recently, Booher et al. reported that lung cells exposed in culture to ozone concentrations as low as 0.3 ppm demonstrated an inhibition in growth that was proportional to the ozone concentration. 

Examine the cells under the microscope to ensure that all the fibroblasts are attached to the tissue culture dishes (Fig. la). Wash the layer of fibroblasts twice with 5 ml of pre-warmed PBS. Seed 2x10 CLL cells (ratio 10 1 10 CLL cells per NIH3T3 cell Fig. lb) on WT and CD154 N1H3T3 in 5 ml complete RPMI medium supplemented with 10 ng/ml of lL-4 (only in CD154 NIH3T3-coated tissue culture dishes) and incubate for 24 h in a humidified atmosphere of 5% CO at 37°C (see Notes 7 and 8). 

Examine the CLL cells by microscopy. The cells should lie on top of the fibroblasts (on both the WT and CD154 NIH3T3) (Fig. Ic). Shake the tissue culture dishes gently to verify adherence. Adherent CLL cells should remain attached after gently shaking the tissue culture dish. 

Detach the CLL cells by gently pipetting up and down using a 1,000 pi pipette tip. Rotate the dishes while pipetting to detach the CLL cells all around the tissue culture dishes. Collect the media containing cells in an appropriate sterile tube. Examine the tissue culture dishes under the microscope to ensure that all the CLL cells are collected and the layer of fibroblasts is still intact (see Note 9) (Fig. Id). 

WT N1H3T3 cells are more prone to detach than CD154 N1H3T3, so detaching CLL cells from the WT N1H3T3 cells requires extra care. If discontinuities or ruptures in the layer of fibroblasts are noticed, CLL cells may be seeded at this step in 60 mm culture dishes for 2 h to allow possible NIH3T3 cells present in the sample to reattach on the surface of the tissue culture dishes. The tissue culture dishes are then incubated in a humidified atmosphere of 5% CO2 at 37°C. Afrer incubation, examine the tissue culture dishes for the presence of adherent cells and carefully collect the CLL cells in an appropriate sterile tube. 

Compounds tested in a more physiological 3-D environment show increased predictability of in vivo responses and help to span the gap between 2-D tissue culture and animal models. There is also evidence that 3-D cultures can identify active compounds that would fail to show their potential in 2-D (56, 57). The complexity of 3-D cultures can be increased by the addition of one or more cell types (fibroblasts, endothelial cells) and/or culture with different media, substrates, or oxygenation conditions. However, a disadvantage is that there is still a lack of simple, standardized, and reliable 3-D protocols that allow their incorporation into the pre-clinical high-throughput validation and drug evaluation process, although there have been several advances in this area in recent years (30, 58-61). 

Fig. 2. Effect of serum concentration on the attachment and spreading of BHK-21 cells onto TCP2 surface. BHK-21 cells were seeded in media containing the indicated concentrations of intact serum (open squares), Fn-depleted serum (triangles). Vn-depleted serum (circles), or serum-free medium alone (the single closed square) and the attachment panel (A) and spreading panel (B) of the cells were determined after 90 min culture on TCP (panel (A, B)) Mean SEM. (Reproduced from J. Biomed. Mater. Res. [Ref. 11 Role of serum vitronection and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene] through the courtesy of John Wiley Sons, Inc.) BHK-21 Fibroblast cell lines from Baby Hamster Kidney 2 Similar results on Primaria are also presented in [Ref 11]...

Adhesion of mononuclear cells from human bone marrow aspirates (BM-MNC) to tissue culture plastic and removal of nonadherent cells during the first days of culture selects for a population of proliferating spindle-shaped fibroblast-like non-... 

Isolated animal cells in tissue culture, no matter how highly differentiated, tend to revert quickly to one of three basic types known as epitheliocytes, mechanocytes, and amebocytes. Epitheliocytes are closely adherent cells derived from epithelial tissues and thought to be related in their origins to the two surface layers of the embryonic blastula. Mechanocytes, often called fibroblasts or fibrocytes, are derived from muscle, supporting, or connective tissue. Like the amebocytes, they arise from embryonic mesenchymal tissue cells that have migrated inward from the lower side of the blastula (Chapter 32). Neurons, neuroglia, and lymphocytes are additional distinct cell types. 

The culture of embryonic fibroblasts is used to obtain enough cells to perform prenatal diagnosis of inherited metabolic diseases (Box 1-D). Tissue culture is easiest with embryonic or cancer cells, but many other tissues can be propagated. However, the cells that grow best and which can be propogated indefinitely are not entirely normal the well-known HeLa strain of human cancer cells which was widely grown for many years throughout the world contains 70-80 chromosomes per cell compared with the normal 46. 

Beckman et al. (28) have studied the electrophoretic separation of the acid phosphatase activity in tissue extracts on starch gel at pH 8. They described four electrophoretic bands A, B, C, and D. Table IV (28) shows the distribution of activity in different organ extracts. The ABD pattern predominated in kidney BD in liver, intestine, heart, and skeletal muscle B in skin and D in pancreas. The C component was present in a large number of placentae but not in other adult organs. All four electrophoretic components were inhibited by d-(- -)-tartrate A contained sialic acid, D had a lower pH optimum and was more heat resistant than A, B, and C. Components C and D showed parallel electrophoretic behavior. In human skin fibroblasts grown in tissue culture, the acid phosphatase was generally high and the most common pattern was BD. Almost every culture showed some activity. The BD... 

Explain why fibroblasts from a patient with I-cell disease secrete lysosomal enzymes when grown in tissue culture. 

Figure 1. Fibroblast cells as seen by stereoscan microscopy Left Surface of cell in tissue culture (X 18,000)...

Isolation and Quantitation of Glycosphinqolipids from Cultured Fibroblasts, Plasma Lipoproteins and Tissue Culture Medium... 

SERCA2a is expressed predominantly in the heart, in slow twitch skeletal muscle and in the brain, where it represents the main SERCA isoform. In contrast, SERCA2b is a house-keeping isoform, ubiquitously expressed in smooth muscle and non-muscle tissue (Wuytack et al., 2002). Although both isoforms are detectable in keratinocytes and dermal fibroblasts in culture, SERCA2b is the major isoform expressed in the epidermis from adult skin sections. SERCA2c is expressed in epithelial, mesenchymal and hematopoietic cell lines, and in monocytes (Table 1). 

Specifically, the data reviewed in Chapters 12 and 13 indicate that LCM are rapidly removed from the circulation by the tumor the maximum accumulation of LCM in the tumor area occurs within the first 30 min after administration (ref. 531). These rapid kinetics for LCM uptake are quite consistent with the well-documented kinetics long-known for the LDL receptor-mediated endocytic pathway (ref. 616). For example, Goldstein et al. (ref. 643) reported that LDL-ferritin bound to coated pits at 4°C is rapidly internalized when fibroblasts in tissue culture are warmed to 37°C. In this uptake process, the coated pits invaginate to form coated endocytic vesicles. After 5 to 10 min at 37°C, LDL-ferritin is observed in lysosomes as the result of their fusion with the incoming coated vesicles (ref. 643). The rapid sequence of events visualized in electron micrographs precisely parallels biochemically-derived data on the rapid uptake and degradation of radiolabeled LDL (ref. 644,645). 

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