The total amount of protein in the precipitate was determined by the standard Lowry procedure. The amount of nidogen and entactin in the gel was related to the total amount of material present in the K band of laminin by scanning negatives of photographs of the gels in a Helena densitometer Quick Scan Model, Helena Lab Corp. Entactin and nidogen were identified based on their migration in SDS gels and cross reactivity in Western blot analyses with suitable antibodies.
Type IV collagen in the gel was quantitated using 14 C-labeled type IV collagen and heparan sulfate proteoglycan was quantitated using 35 S-sulfate labeled material of known specific activities in separate but parallel experiments.
Rotary Shadowing - The 2. For rotary shadowing, the mixture was sprayed onto mica, shadowed with platinum-palladium, carbon coated, and examined in a JEOL C electron microscope. Ultrastructure of Reconstituted Components - The gel was prepared essentially as described above.
Briefly, 0. The gel was isolated by centrifugation and then fixed in 2. Thin sections of rat kidney tubule basement membranes were obtained as described by Laurie et al, Am. After one week, the cells were photographed. The assembly of basement membrane components was analyzed using purified basement membrane components as well as unfractionated extracts of basement membrane. The components of the gel were isolated by centrifugation and examined by SDS gel electrophoresis. As shown in FIG.
Heparan sulfate proteoglycan also caused increasing amounts of basement membrane components to precipitate FIG. Separation by gel electrophoresis and quantitation of the major components in the gel indicated that constant ratios of laminin, entactin, and nidogen are obtained in the presence of added type IV collagen FIG.
The smaller chain of laminin co-electrophoresed with the chains of type I. V collagen and prevented its visualization in the SDS gel. To estimate the amount of type IV collagen in the gel, 3 H-labeled type IV collagen of known specific activity was used and the amount of 3H-label in the precipitate was used as a measure of type IV collagen. Likewise, the heparan sulfate proteoglycan cannot be visualized in the gels and 35 S-labeled heparan sulfate proteoglycan was used.
In contrast, supplementation of the extract with eiher type I collagen, fibronectin or heparin FIG. Removal of the protein core of the proteoglycan by incubation overnight with 0. Under physiological conditions, the gelation process is complete within 20 minutes FIG. The stability of the gel to dissolutioh was examined by using various solvents. The gel was not dissolved by cold aqueous salt but was partially dissolved by acidic solutions Table 1 and completely dissolved in guanidine or in urea solutions. This suggests that the components are linked by relatively strong non-covalent bonds.
When the guanidine-dissolved gel was dialyzed against physiological buffers and warmed in the presence of type IV collagen, gel-like structures were reconstituted. This process could be repeated several times with similar proportions of laminin, nidogen, and entactin being deposited at each step as determined by SDS polyacrylamide gels FIG. In the presence of added type IV collagen, reformation of the gel occurred more rapidly and greater amounts of the components were deposited. Determination was also made whether soluble complexes of basement components existed.
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When the urea extract was dialyzed free of urea and passed over a Sepharose 4B column in 0. When the material in the major included peak was pooled and rerun over the same molecular sieve column in 4M guanidine dissociative conditions , these components separated in the manner expected from their molecular weights FIGS. These results indicated that there are strong but noncovalent bonds joining laminin, nidogen, and entactin in the complex. Rotary shadowing electron microscopy of the major included peak material confirmed the presence of soluble complexes FIG.
The complexes involved the large proteoglycan which appears as a large globule due to collapse of the heparan sulfate side chains in this kind of preparation surrounded by several laminin molecules. The nidogen and entactin molecules could not be distinguished but are known to be in the complexes from SDS polyacrylamide gels FIG. The ultrastructure of the reconstituted basement membrane either with or without type IV collagen and heparan sulfate proteoglycan was also examined. In the absence of added type IV collagen and heparan sulfate proteoglycan, the gel consisted of numerous widely separated thin, filamentous aggregates FIG.
The individual segments of the network had an average width similar to that of the lamina densa of kidney tubule basement membrane FIG. However, unlike native basement membranes in which lamina densa-like layers are arranged in parallel, such as for example the PYS tumor basement membranes Martinez-Hernandez et al, Lab Invest. At very high power in the electron microscope, each segment could be resolved into 5 nm cords as previously described in other basement membranes Inoue et al, supra, Laurie et al, J.
The matrigel reconstituted basement membrane was used to coat the surfaces of bacteriological petri dishes and tested as a substrate for the growth and differentiation of a variety of cells at different laboratories. Melanoma cells B16C3 showed considerable differences in morphology when grown on the basement membrane gel as compared to tissue culture surfaces FIG. Further, there was a much earlier and more extensive pigmentation of the cells on this substrate. Studies of other cells showed that endothelial cells formed tube-like structures on the gel and that hepatocytes survived longer on basement membrane gel substrates than on tissue culture plates or on type I collagen.
In vivo, the basement membrane gel was found to promote peripheral nerve regeneration Madison et al, Such studies indicate that the reconstituted basement membrane is a biologically active substrate which induces diverse cellular responses. Since it can support cell adhesion, growth and differentiation beyond that known for the individual components, without being bound to any theory it is postulated that the reconstituted basement membrane gel contains these molecules in a unique and active conformation.
Use a graduated cylinder for 1 liter. Inside of bag is sterile. Render the outside of the bag sterile with alcohol, rinse hemostat and scissors in alcohol and empty bags in sterile hood into sterile containers. Aliquot as needed.
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Collagen IV can be optionally added at this stage to the liquid phase in an amount ranging from about 0. The thicker gels have been found to be more durable. Immediately cool. For a 35 mm petri dish, use less than 1 ml of the extract. Spread it thin. To use the gel as a cell culture substratum, add about 3 ml of suitable growth medium on top of the polymerized gel obtained from step 18 and inoculate the medium with the dispersions of the cells which are desired to be grown.
Of course, the growth medium to be used will depend on the type of the cell which is desired to be grown; specific standard growth medium and conditions e. Co 2 concentration, temperature, pH and the like for different types of cells being well known in the art. An alternate procedure for promoting the growth of some cell types is to inoculate or disperse the cells in the cold liquid extract just before polymerization in step 18 and then proceed with polymerization and subsequent steps the same as described in step For example, hair follicle, sertoli cells and the like are apt to be better cultured if first dispersed in the liquid phase prior to polymerization whereas epithelial cells, exocrine acinar cells, sciatic nerve cell, spinal cord neuron, thyroid organ culture, and the like are better cultured on top of the polymerized gel.
Extracts comparable in composition and in biological activity can also be obtained from human placenta using a process similar to that used for the EHS mouse tumor described herein. However, since placenta is not composed of pure basement membrane like the EHS mouse tumor. Both the 0. The bound material is eluted with 1. The placental extract before and after heparin sepharose chromatography were compared for the purity of the extract.
To do so, the 0. The samples were then stained with Coomassie blue for a profile on the protein content and immunoreacted with anti-laminin antibodies after transfer to nitrocellulose. These results demonstrated that laminin, the major component of basement membranes, as of the mouse basement membrane preparation described herein, is present in the placenta extracts in intact form as clearly demonstrated after heparin affinity chromatography FIG. The biological activity of this material on neurite outgrowth was tested using NG neuroblastoma plus glioma hybrid cells in culture.
kryolanjerusalem.com/modules/software/2897.php These cells respond rapidly within 2 hours to the extracts as well as to the heparin bound material by sending out long neuritic processes FIG. The material to be tested is added in Eagle's minimal essential medium lacking serum or some other culture medium along with freshly dissociated cells.
After two hours on tissue culture plastic, extended processes are observed in the cells exposed to the placental material. Thus, the placenta materials have comparable activity to the murine tumor material in stimulating neurite process development. In order for all tumor cells to metastasize, they must enter the blood stream and then exit from it to grow at a distant site.
Tumor cells must therefore adhere to, degrade, and migrate through endothelial basement membranes in order to metastasize. These steps are critical in tumor cell metastasis. A unique in vitro assay to measure these critical steps in the invasion process has now been devised. The assay is fast, quantitative, reproducible, and distinguishes between nonmetastatic and metastatic cells.
This assay employs the murine reconstituted basement membrane described herein.
A porous filter Nucleopore is placed inside a blind well Boyden chamber. The lower compartment contains an attractant such as fibroblast conditioned medium or laminin. During this time, the invasive cells adhere to the matrix, degrade the matrix, and migrate through the matrix and the porous holes in the filter. This process is diagrammatically shown in FIG. The number of cells which have invaded the matrix can be quantitated on the lower side of the filter, for example by direct counting in a microscope after the cells have been stained with DifQuick Harelco.
Alternatively, if the cells are radiolabeled, they can be measured directly in a scintillation counter. It was observed that cells which are known to be non-metastatic, in vivo, do not invade the matrix, i. Whereas, cells which are known to be metastatic in vivo invade the matrix, i. More than 10 murine and human tumor cell lines of known metastatic potential have been tested and it was found that there is a direct relationship between the ability of the cells to adhere to, degrade, and migrate through the reconstituted murine basement membrane and their metastatic potential Table II.
Highly invasive tumor cells can also be selected for and obtained in pure form based on their ability to adhere, degrade, and migrate through the reconstituted basement membrane. Here the murine basement membrane extract is placed on a tissue culture dish 0. The cells are plated in a sterile manner in complete culture medium as required for the growth of the specific cells.
After two days, the invasive cells attach to, degrade, and migrate through the matrix to the surface of the plastic dish where they are concentrated. This is shown in FIG. The invasive cells on the plastic surface can be recovered after removal of the reconstituted basement membrane gel. It was found that non-metastatic cells are unable to complete this process and very few, if any, cells are observed in the Matrigel or on the plastic dish after 2 days Table III.
Metastatic cells are able to adhere to, degrade, and migrate through the matrix to the surface of the culture dish in quantity.