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Correspondence to: Kevin J. McCarthy, Louisiana State U. Health Science Center, Dept. of Pathology, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932. E-mail: kmccar2@lsuhsc.edu
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Summary |
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L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions. (J Histochem Cytochem 50:15371541, 2002)
Key Words: extracellular matrix, basal lamina, basement membrane, critical assembly concentration, dialysis membrane
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Introduction |
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Basement membranes (BMs) are specialized extracellular matrices (ECMs) which in part separate epithelial cells from the underlying stroma and surround smooth muscle, nerve, and fat cells (
L-2 cells are a tumor cell line derived from parietal endoderm cells which produce Reichert's membrane, a basement membrane transiently present during rodent development (
To test our hypothesis, we limited the diffusion of secreted products to the immediate pericellular area while permitting the cells access to nutrients necessary for survival. Our technique uses a semipermeable membrane to limit diffusion of secreted products in a 24-well plate format. Our observations indicate that L-2 cells, which do not assemble a significant matrix under routine cell culture conditions, can be induced to assemble a robust BM matrix in 24 hr when the diffusion of secreted products is limited by a semipermeable membrane.
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Materials and Methods |
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Reagents and Supplies
The L-2 cells were a kind gift from Ulla Wewer (Copenhagen, Denmark). The heparan sulfate proteoglycan (perlecan) antibody C17 and the s-laminin (ß2 chain) antibody C4 were originally developed by Dr. Joshua Sanes (
Cell Culture Conditions
L-2 cells were maintained as previously described (
Immunohistochemistry and Digital Microscopy
After fixation the cells were washed three times for 10 min with PBS. The cells were blocked for 20 min with 1% BSA in PBS, followed by another PBS wash three times for 10 min. For immunostaining, the anti-laminin polyclonal antiserum was used at a 1:100 dilution in PBS. MAb supernatants were applied directly to the cells without dilution for 45 min, followed by a PBS wash three times for 10 min. The labeled secondary antiserum was diluted at 1:100 in PBS and applied to the cells for 45 min. Then the cells were washed three times for 10 min with PBS and then directly imaged within the wells to maintain the 3D dimensional organization of the assembled ECMs. Digital images of the specimens were acquired using an Olympus IX-70 microscope equipped with epifluorescent illumination optics and appropriate filters. The microscope was interfaced to a SenSys camera (Roper Industries; Tucson, AZ) whose signal was ported to a PowerMacintosh 9600 hosting the imaging acquistion/analysis software IPLab spectrum (Scanalytics; Fairfax, VA).
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Results |
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When the diffusion of secreted products from L-2 cells was limited by the application of a dialysis membrane (MWCO 1214,000), a robust matrix containing laminin was assembled within 24 hr (Fig 1B) and continued to be formed for 48 (Fig 1D) and 72 hr (data not shown). L-2 cells grown for 24 or 48 hr without a dialysis membrane secreted laminin but did not develop an organized laminin matrix (Fig 1A and Fig 1C). In those wells the laminin staining had a punctate pattern. Similar patterns of matrix deposition were seen in wells immunostained for type IV collagen that contained L-2 cells grown with (Fig 1F) or without (Fig 1E) a diffusion-limiting membrane. The antibodies directed against perlecan (Fig 1G) or BM-CSPG (Fig 1H) also showed the development of pericellular matrices in cultures using the diffusion-limiting membrane. Thus, the L-2 cells assemble a BL matrix that contains at least one isoform of several common BL-associated molecules. Interestingly, the pericellular matrices immunostained with the MAb directed against the ß2 laminin chain were negative for L-2 cell cultures under both growth conditions (data not shown).
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Discussion |
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In this report we have described a technique using a semipermeable membrane to induce the assembly of a basal lamina in a cell culture system within a 24 hr period.
We use the term BL to describe the pericellular matrices assembled by L-2 cells. In the literature, the term BM is often used by many in an interchangable manner with the term basal lamina. However, in the strictest sense, the term "basement membrane" refers to the complex ECM that underlies, for example, epithelia. Its constituent parts include the BL and the reticular lamina (
Although BLs have been extensively studied in animal model systems, in vitro modeling of these structures in some cell types is often difficult. Some cells, e.g., epithelial cells, readily produce a BM in culture. In light of the CAC hypothesis, this may be due to the fact that polarized epithelia will directionally/vectorially secrete BL components to their basal surface, where diffusion of these components into the surrounding medium is directly limited by the intact cellular monolayer. With other cell types, assembly occurs only after prolonged culture, or may require co-culture with a nurse cell, which can complicate in vitro modeling (
We believe that the using the CAC method has several advantages. (a) It allows cells to rapidly develop a true native BL matrix, especially those cells such as L-2 cells that appear to secrete ECM proteins from their entire cell surfaces in a non-vectorial fashion. (b) Once cells have developed their own native matrix, the culture is readily accessible for further in vitro studies with or without the diffusion-limiting membrane. (c) The CAC method allows the study of the development of a matrix by a cell line that may not readily assemble a matrix in vitro. (d) The CAC method could be applied to any situation in which limiting the diffusion of secreted products to mimic in vivo concentrations would be useful, such as studying the effect of natively secreted growth factors or chemokines.
This report further demonstrates the importance of limiting secreted protein diffusion to enhance local pericellular protein concentrations to promote matrix assembly in a cell culture system. In earlier reports Yurchenko and co-workers demonstrated that BL assembly can be recapitulated in vitro using purified basement membrane components (
Although the evidence of a critical concentration of components necessary for the assembly of a matrix has been portrayed in this report, there are other avenues that will guide future work. If the concept of CAC is induced by this apparatus, one would predict that increasing the MWCO of the dialysis membrane would result in an inverse deposition of matrix. Similarly, by varying the amount of medium beneath the membrane directly in contact with the cells, the CAC hypothesis would predict that increasing the volume of medium bathing the cells would result in a decrease in the extent of assembly of the pericellular matrix.
In summary, we have developed a technique that uses a dialysis membrane to limit the diffusion of proteins secreted from L-2 cells to induce the assembly of a robust matrix within 24 hr. We propose that the membrane has caused a critical association complex (CAC) to form, concentrating components necessary for matrix assembly.
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Acknowledgments |
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Supported by a research grant 1-RO1-DK48055 (KJM).
This work was submitted in partial fulfillment of the PhD degree (MEL) for the Louisiana State University Health Sciences Center, Shreveport, LA.
Received for publication June 4, 2001; accepted May 1, 2002.
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