A General and Reliable Method for Obtaining High-Yield Metaphasic Preparations from Adherent Cell Lines: Rapid Verification of Cell Chromosomal Content
In vitro studies are becoming more and more frequent in numerous domains of cell biology. For such purposes, a great variety of cell lines have been established either from normal tissues or, more frequently, from tumours. Very few of these cells present a normal karyotype: they are generally hyperdiploid and often contain rearranged chromosomes. Moreover, depending on the length of time in culture and on the culture conditions, populations of these cell lines can change and drift. Therefore, some general culture rules have to be respected (Ian Freshney, 1987), and it is highly recommended to avoid working with cells maintained in culture for a large number of generations.
Because of possible drift, cell lines need to be followed and controlled, with one fundamental control being the stability of their chromosomal content. Unfortunately, such control is not systematic. Moreover, in the case of new cell lines, it is frequent that their phenotype is described in detail with little or no information on their karyotype. It should be noted that even if karyotyping methods have long been described in the literature (for the history of human cytogenetics, see Jeening Lawce and Brown, 1997), it is not always easy to obtain metaphasic preparations from some cell lines. We were confronted with such a situation for some hybrid lines, in particular polarized rat hepatoma-human fibroblast WIF clones (Cassio et al., 1991; Shanks et al., 1994). This article describes a new method for obtaining, at a high yield, metaphasic preparations from delicate cell lines. This method is easy and reliable and has been applied successfully by many people to more than 50 different cell lines from different species.
A. Principles of the Method
This method is a combination of the two methods described by Worton and Duff (1979), the suspension method generally used for nonadherent and adherent cells and the "in situ" method that has been specifically developped for adherent cells (Cox et al., 1974; Peakman et al., 1977). When we tried to prepare metaphases from the well-polarized hybrid cell line WIF-B (Shanks et al., 1994), we only succeeded with the "'in situ" one. We performed many assays using the suspension method and they all failed: from two to three petri dishes containing 106 cells/dish in exponential phase, we obtained, at the best, a dozen metaphases (yield <10-5). In contrast, with the in situ method, the metaphase yield was very good and attained 1.5% of the total cell number (the maximal expected value for cells with a generation time of 2.5 days).
In the suspension method, mitoses are detached (mechanically or by proteolysis), whereas in the in situ method, mitoses stay in place. As polarized WIF-B cells have a highly differentiated plasma membrane, organized in different domains, we hypothesized that mitotic cells from this line are very fragile and are lost during or after detachment (may be during centrifugation). Therefore, to isolate metaphases from this line, a new method was developped that avoids cell detachment, at least during the early steps. The first steps of this method (metaphase arrest, hypotonic swelling, and first fixation) are performed in situ and it is only after the first fixation that cells are detached.
B. Advantages of the Method
This method presents several advantages. First, it is applicable to every adherent cell line and does not depend on the adherent properties of the mitosis, as all mitoses (floating and adherent ones) are collected. Second, as with the in situ method, the yield in metaphases is very good (Fig. 1A) and therefore the metaphasic preparations are representative of the whole cell population. Third, this method requires fewer cells and is less wasteful in cells than the suspension method, an advantage for slow-growing cell lines where mitotic cells are rare or for cells where the results of karyotyping are required as soon as possible. Finally, as with the suspension method, the quality of the metaphase spreading is good (Fig. 1B) and can be adjusted, whereas with the in situ method, which allows only one attempt per culture, the spreading cannot be controlled and is very sensitive to cell overcrowding. Moreover, the chromosomes obtained by this new method are quite suitable for G banding, Giemsa 11 staining (Buys et al., 1984) (Fig. 1C), and fluorescent in situ hybridization (FISH) (Fig. 1D).
II. MATERIALS AND INSTRUMENTATION
Growth medium (available from local suppliers)
Colcemid (10µg/ml; GIBCO, Cat. No. 2465)
Gurr buffer tablets, pH 6.8 (BDH, Cat. No. 33199)
Giemsa solution (Merck, Cat. No. 1.09204.0100)
10-cm tissue culture dishes (Falcon, Cat. No. 3003)
15-ml tubes (Falcon, Cat. No. 352099)
Precleaned, ground edge, microslides (ESCO, Cat. No. 2951R)
A low-speed centrifuge with a swinging-backet rotor for 15-ml tubes (as the IEC clinical centrifuge) is needed for harvesting cells, and a phase-contrast microscope is needed for examining cells and slides.
III. PROCEDURES A. Cleaning and Frosting of Microscope Slides
B. Optimization of Growth
Grow cells in 10-cm Petri dishes in appropriate medium so that cells will be in midexponential phase with many mitoses on the day of harvest. Renew the dishes with 10ml of medium the day before the harvest.
C. Metaphase Arrest
Add 0.2ml of colcemid for 1 h at 37°C to the dish containing the maximal number of mitotic cells. The other dishes can be used later if necessary.
D. Hypotonic Swelling
Note: The swelling of the cells can be verified by examination under a microscope.
E. Fixation Steps
F. Spreading and Air Drying
Spreading is done by air drying onto frozen slides.
As the present technique was developed to verify the cell chromosomal content, we use Giemsa "solid staining" (Worton and Duff, 1979), which gives uniform staining of the chromosomes and makes it easy to count them. However, other staining methods can be applied (Fig. 1).
Using this method, metaphasic preparations from of a panel of well-known and frequently used lines, including polarized lines (Caco-2, MDCK, HT-29) and hepatic ones, were isolated and analyzed (Table I). Some lines (BW1-J, cl l-D, HT-29) displayed a chromosomal content similar or very near to that published previously, but the mean number of chromosomes, as the range, of other lines (Caco-2, HeLa) differed greatly from those published. Morever, in some cases, big differences were observed from the same line obtained from different laboratories. This was the case for HeLa cells (Fig. 2) and, to a lesser extent, for MDCK. In this latter case, one population over the three tested was very heterogeneous and greatly differed from the two others. Although both L and HeLa lines were established a long time ago (in 1940 and 1951, respectively) and thus cultured for a very large number of generations, the first line seems to be very stable, whereas the second one has considerable drift.
I thank C. Delagebeaudeuf for pushing me to develop this method, C. Hamon-Benais, and V. Bender for the illustrations, and L. Sperling for careful reading of the manuscript. This work was supported in part by the Curie Institute (PIC Signalisation Cellulaire Grant 914), CNRS, and INSERM (contrat Prisme 98-09).
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