CA1266603A - Membrane invasion culture system - Google Patents

Abstract

MEMBRANE INVASION CULTURE SYSTEM

ABSTRACT OF THE DISCLOSURE
A cell culture system for performing cell invasion assays comprises a base plate and a top plate.
The base plate includes a plurality of wells formed in one face thereof, while the top plate includes a plurality of apertures arranged in substantially the same pattern as the wells in the base plate. By placing a test membrane between the plates and securing them together, a plurality of test receptacles are formed, each receptacle having an upper chamber and a lower chamber separated by the test membrane.
Invasion assays are performed by filling the lower chamber with a suitable culture medium and seeding the cells to be tested in the upper chamber.

Description

~66~i~3 MEMBRANE INVASION CULTURE SYSTEM

BACKGROUND OF THE INVENTION

Malignant tumors are comprised of a heteroge-neous population of cells, only a small subpopulation of which have the capability to metastasize. The ability of tumor cells to metastasize has been linked to the ability to penetrate various biological membranes in the body. Thus, the study of tumor cell invasiveness is important in elucidating the mechanism of cancer cell metastasis and may prove beneficial in devising therapeutic regimes for individual patients.

A number of in vitro models for studying mem-brane invasion have been reported. One such system designated the vascular endothelial cell monolayer (Kramer and Nicolson (1979) Proc. Natl. Acad. Sai. USA
76:5704-5708) is limited in that the cells are cultured on plastic and the tumor cell/endothelial cell interac-tion can only be examined in one dimension. Anotheravailable system is the chick chorioallantoic membrane model described by Easty and Easty ~1976) in: "Organ Culture and Biomedical Research, n Balls and Monnickendam (eds.), Cambridge University Press, Cambridge, pp. 379-392. This system suffers both from the fragility of the membrane and from possible tissue incompatibility when human tumor cells are assayed.
Additio~al tissue invasion models include monolayers of vascular endothelial cells combined with smooth muscle cell multilayers (Jones and DeClercg (1980) Cancer Res.
40:3222-3227); a perfused canine vein system IPoste e*
al. (1980) Cancer Res. 40:1636-1644); a human decidua ~

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~26~i6~3 graviditates model (Schleich et al. (1976) J. Natl.
Cancer Inst. 56:221-225); and urinary bladder membranes (Hart (1979) Amer. J. Pathol. 97:587-600). A
particular system for immobilizing a biological membrane in a receptacle for performing membrane invasion assays is described by Russo et al. (1982) in:
"Tumor Invasion and Metastasis," Liotta and Hart (eds.), pp. 173-187, Martinus Nijhoff Publishers, The Hague, Netherlands. See page 177 where the particular system is illustrated. The system is further described in ~.S. Patent No. 4,446,234. See also, Liotta et al.
(1981) Cancer Res. 41:4629-4636; Liotta et al. (1983) Lab. Invst. 49:636-649; Terranova et al. (1982) Cancer Res. 42:2265-2269; Russo et ~1. (1981) J. Cell Biol.
91:459-467; Tchao et al. (1980) in: "Metastasis, Clinical and Experimental Aspects," Hellman et al.
(eds.), Martinus Nijhoff Publishers, The Hague, Nether-lands; Kramer et al. (1982) in "Interaction of Platelets and Tumor Cells," Alan R. Liss, Inc., New York, New York, pp. 333-351; and Scheich et al. (1981) Arch. Geschwulstforsch 51:40-44.

The present invention provides a novel system and method for performing cell invasion assays for assessing the ability of living cells to penetrate bio-logical or synthetic membranes. The method employs a novel cell culture system which can utilize a single prepared membrane to define a plurality of test receptacles. The culture system comprises a base plate having a plurality of wells formed in one face thereof, and a top plate having a plurality of apertures formed therethrough in a pattern corresponding to that of the wells in the base plate. Means are provided for securing the top plate to the base plate so that the apertures are aligned with the wells to form the test .
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receptacles. By placing a prepared biological or synthetic membrane between the base plate and the top plate, the culture system is assembled with each of the test receptacles being partitioned into upper and lower chambers. In the preferred embodiment, ports are provided to allow access to the lower chamber without disassembly of the culture system.
Using the cell culture system just described, cell invasion assays may be performed by first filling the wells in t,he base plate with a suitable culture medium for the cells being tested. The desired membrane is then prepared. For example, an amniotic membrane may be prepared from a human placenta and secured between the base plate and top plate. The cells of interest, typically tumor cells or cells suspected of being neoplastic, are then introduced to the upper chamber of a test receptacle and allowed to incubate for a predetermined period of time. Cell invasiveness can be measured by taking samples of the culture medium from the lower chamber of the test receptacle to determine whether any cells have migrated through the membrane. At the end of the assay, the membrane can be removed from the culture system and examined for evidence of cell penetration. 'Such assays are useful both for studying the mechanism of cell invasion and for determining the chemotherapeutic effectiveness of drugs against individual patient tumors. The latter allows the clinician to identify the most effective drugs for treatment of each tumor in question.
The culture system of the present invention provides a number of advantages over the prior art. By providing an easily separable base plate and top plate, the insertion of the membrane into the system is greatly facilitated. The membrane may be simply placed over the base plate and, after securing the top plate thereon, the excess membrane may be trimmed from the , _. .

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~Z6Çi6~3 edges. Such an approach minimizes handling of the membrane and reduces the risk of damage thereto.
Moreover, the relatively large contact area between the top and bottom plates holds the membrane firmly in place while avoiding stress on the membrane which can lead to tears and other damage. Furthermore, by providing a plurality of test receptacles in the system, the total labor required per unit test is greatly decreased.

In the drawings:
Fig. 1 is a perspective view of the cell cul-ture system o~ the present invention.
Fig. 2 is a sectional view illustrating a single test receptacle taken along line 2-2 of Fig. 1.
Fig. 3 is a sectional view illustrating an alignment peg taken along line 3-3 of Fig. 1.

Referring to Fig. 1, a particular cell culture system 10 employing the concepts of the present 20 invention is illustrated. The cell culture system 10 comprises a base plate 12 and a top plate 14 which are held together by a central screw 16. Alternative means for firmly securing the plates 12 and 14 together, such as clamps, clasps, and the like, would also be 25 suitable. The culture system 10 includes a plurality of individual test receptacles 18. Referring to Fig.

2, each test receptacle is defined by a well 20 formed in the base plate 12 and an aperture 22 formed in the top plate 14. The well 20 and aperture 22 will 30 typically have the same cross-section profile and wil~
be hori~ontally aligned when the top plate 14 is secured to the bottom plate 12. The dimensions of the receptacles 18 are not critical, although each of the receptacles will usually have identical dimensions, 35 typically being circular with a diameter between about .. : .

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~26/~6~3 0.5 and 2.0 centimeters and having a depth in the range from about 1 to 3 centimeters. Alignment pins 24 (Fig.

3) are provided to assure that the wells 20 and apertures 22 are properly aligned when the plates 12 and 14 are placed together. Conveniently, the alignment pins 24 will be extended above the surface of the top plate 14 to provide a post 25 for supporting a protective cover (not illustrated) over the open end of the receptacles 18.
To complete the cell culture system, a test membrane 26 is secured between the base plate 12 and top plate 1~. The result is that the membrane 26 partitions each test receptacle into a lower chamber defined by the test well 20 and an upper chamber defined by the aperture 22. Thus, wihen cells are seeded into the upper chamber 22, they can only penetrate into the lower chamber 20 by migrating across the test membrane 26. Conveniently, a port 28 is provided to allow access to the lower chamber 20 from the outside while the membrane 26 remains in place.
The port 28 threadably receives a plug 30 to prevent loss of the culture medium from the lower compartment 20 when samples are not being taken.
The materials of construction for the culture system are not critical. Suitable materials include glass, stainless steel, ceramics, aluminum, and plastics such as polyvinylchloride, acrylic resin polymers, polypropylene, polyethylene, polycarbonate, and tetrafluoroethylene fluorocarbon polymers, and the like. Depending on the materials of construction, the method of fabrication may vary. Plastics will typically be injection molded, although both plastics and metals may be machined from sheets of material. It is desirable that the cell culture system be sterilizable by heat in an autoclave, so that high temperature plastics are preferred. Alternatively, the system may be sterilized by a gas sterilant, such as -:'~

~26~;6~33 ethylene oxide. Also, it is possible that certain metals may be toxic to the particular cells under investigation, and such metals should be avoided.
A wide variety of both natural and synthetic membranes may~be utilized in the cell culture system of the present invention. Natural biological membranes will usually be of the greatest interest, and virtually all of the biological membranes disclosed in the prior art may be adapted for use, as well as other membranes which have not heretofore been utilized. Suitable bio-logical membranes include human amniotic basement mem-brane, human pericardium membrane, human peritoneum membrane, chick chorioallantoic membranes, inner egg shell membranes, and the like. Alternatively, synthetic membrane barriers can be utilized, such as a synthetic matrix formed from type I collagen, and other gelatinous semipermeable synthetic membranes may find use. Also, paper like membranes, such as millipore filter paper, may be used in the present invention.
Particularly preferred are amniotic basement membranes prepared in the manner described in the experimental section hereinafter. Such membranes are preferred both because of their histocompatibility with human tumor cells and their widespread availability from hospital maternity units. In some cases, it may be desirable to utilize a synthetic support matrix to strengthen fragile biological membranes or synthetic membranes composed from cultured cells.
Assays utilizing the cell culture system of the present invention will generally be performed as follows. After sterilizing the system components, typ-ically in an autoclave or by use of a gas sterilant, a desired culture medium is placed in the wells 20 formed in the base plate 12. The wells 20 will be completely filled so that they will contact the lower surface of the membrane 26 when it is in place. The membrane 26 will then be prepared and placed over the upper facè of .... .

~26~ )3 the base plate 12. Top plate 14 is then aligned so that alignment pins 24 penetrate the membrane 26 and are received in the corresponding holes in the base plate 12. The top plate is then secured by screw 16.
The cells to be tested are then seeded into the upper compartment 22 in a suitable culture medium, and are incubated for a preselected period of time. While the cells are incubating, samples of the culture medium may be removed from the lower compartment 20 through the associated access port 28. After removing a sample, additional culture medium should be reintroduced to maintain the proper level of medium in the lower com-partment 20. Tests run in each of the test receptacles 18 may be identical or different, and usually controls employing cell lines having known properties will be run in at least some of the wells. A particular advan-tage of the present system is that it allows a plurality of tests to be run simultaneously using a single test membrane 26. In this way, the effect of varying the cells being tested, the culture media, or both, may be studied.
Samples of the culture media taken from the lower compartments both during and after the invasion assay may be tested in a variety of ways. The number of cells which have penetrated the membrane may be counted, typically using a commercial cell counter, such as a Coulter counter. Additionally, the viability of the cells may be tested in a conventional manner, such as a dye exclusion test. The media may also be tested for the presence of various metabolites and other factors which may affect the ability of the cells to penetrate the membrane. After collecting cells from the lower chamber, either through the sampling port 28 or after the membrane 26 has been removed, the collected cells may be recultured for use in further experiments. Such collection in prior art systems (which do not provide an open lower compartment beneath - - ' ''~' ' ~ ' ~
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~L266~i~3 the membrane) has required that the assay system be disassembled and the assay terminated.
The culture system of the present invention may also be utilized in connection with patient therapy. By utilizing cells derived from a patient tumor, the effectiveness of various drugs in inhibiting cell invasiveness may be measured. After culturing the tumor cells, the drugs may be introduced either by pretreating in the culture or by adding them directly to the test receptacles 18 in the cell culture system 10. In some cases, it may be desirable to employ a biological membrane derived from the treated patient, e.g. from the pericardium or peritoneum.
The following experiments are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

MATERIALS AND METHODS
1. Culture ~y~em The culture system employed in the following experiments was essentially as illustrated in Fig. 1.
The base plate and top plate were formed from an acrylic resin polymer, each plate having a length of 10cm, width of 6.2cm, and a thickness of 1.7cm. The wells and apertures formed in the base plate and top plate respectively had a diameter of 1.2cm, and the depth of the well was 1.3cm at its apex.
2. Cell Lines A human melanoma cell line designated 81-46c was obtained from Dr. Frank Meyskens, Jr., University of Arizona Cancer Center, Tucson, Arizona. Tumor cell suspensions were prepared and used in a soft agar clonogenic assay as described by Thomson et al. (1982) 3S Cancer Res. 42:4606-4613. Following the soft agar assay, colonies were plucked from the agar after 14 days, placed in a conical test tube containing Ham's .. ~

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~26~6~3 F-10 media, and mechanically dissociated. Cells were then plated in Falcon flasks with Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) containing 10%
fetal calf serum. Passages 2-5 from the human melanoma cell line were used in this study and were shown to be tumorigenic in athymic nude mice.
Nontumorigenic cell lines were used in this study as a control for tumor cell invasiveness. These consisted of neural crest-derived melanocyte cell lines isolated from chick neural tubes (as described by Loring et al. (1981) Dev. Biol. 82:86-94) and human foreskin fibroblasts obtained from the American Type Culture Laboratory.
3. Preparation of Membrane Invasion Culture System Fresh human placentae were obtained from the Labor and Delivery Unit at the Arizona Health Sciences Center. In each case, the amniotic membrane was gently dissected away from the chorion, washed several times with sterile PBS (phosphate buffered saline, pH 7.4), Fungizone~ (Irvine Scientific, Santa Ana, California), and sterile DMEM before being placed in the culture system described above. The amnion was aseptically interposed between the top and base plates with the epithelial surface facing the top plate. The fastening screw was tightened, and the extra membranous material was trimmed away with a scalpel blade. Prior to the fitting of the membrane in the chamber, the bottom wells were filled with sterile DMEM containing 10%
fetal calf serum.
In order to study the interactions of tumor cells with an acellular basement membrane and underlying stroma, the amniotic epithelium was removed.
In this manner, the collection and counting of penetrating cells was not complicated with an additional cell type and interaction. The amnion was treated with 0.25M ammonium hydroxide for 7 min at room temperature, followed by extensive washing in PBS.

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4. Assay Protocols Tumor cells (81-~6c) or control cells (neural crest or fibroblasts) were pipetted into each well in serum-containing DMEM at a final concentration of loO x 104/ml, and the culture system placed in a humidified incubator at 37C with 5% CO2 and 95% air atmosphere.
After a prescribed period of time, random samples of denuded basement membrane were prepared for scanning and light microscopy in order to check the integrity of the membrane. In addition, all membranes were carefully examined fGr leaks prior to cell seeding by incubating the denuded basement membrane surface for 1 hr with density marker beads (Pharmacia) with a buoyant density of 1.049gm/ml in a percoll gradient containing 0.25M sucrose. If the colored beads, which were the same density as the cells from each cell line, were detected in the bottom wells, the corresponding portions of the membranes were not used in the experiments.
At the completion of preselected time periods, the number of tumor cells that were able to invade the basement membrane and underlying collagenous stroma were counted from the bottom wells via the sampling ports or by the removal of the membrane.
Cells were counted with a Coulter counter or~a hemocytometer, and were subjected to the Trypan Blue Exclusion Test.
Sensitivity of 81-46c cells to Actinomycin D
(Act-D) was also measured. The cells were incubated with O.OOl~g/ml Act-D for the first 24 hr of their in-teraction with the membrane. The Act-D was then removed from each experimental well, and fresh medium was added for the remaining 2 to 3 days.

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., ~. ~ :, ~26~3 RESULTS

Data collected from the invasion assays uti-lizing the cell culture system of the present invention with human melanoma tumor cells ~81-46c), fibroblasts and neural crest-derived melanocytes are presented in Table I. These data clearly demonstrate the invasive-ness of the melanoma cells in contrast to the control cells which are unable to penetrate the membrane.
Twenty-five percent of the total number of 81-46c cells placed on the membrane were able to penetrate the base-ment membrane by day 3. Thirty-two-percent of this number penetrated the membrane by day 4. Control fibroblasts and neural crest-derived melanocytes were not detected in the bottom wells of the cell culture system. The Trypan Blue Exclusion Test was performed with samples of cells from the upper and lower wells of MICS to determine the percent cell viability. The effect of Act-D on the invasion capability of 81-46c melanoma cells is also shown in Table I. Act-D reduced the number of tumor cells that were able to successfully invade the basement membrane by 66% on day 3 and 72% on day 4.

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~;~666~)3 TABLE I
CELLS PENETRATED
DAYS IN NO. IN % VIABILITY
CELL TYPE CULTURE TREATMENT BOTTOM WELLS (TOP WELL/BOTTOM WELL) 81-46c 3 N/A2500 91.0%/89.1~
81-46c 4 N/A3200 87.3%/86.2%
Fibroblasts 3 N/A O 93.0%/ 0.0%
Fibroblasts 4 N/A O 89.2%/ 0.0~
Neural Crest 3 N/A O 84.0%/ 0.0%
Neural Crest 4 N/A 79.2%/ 0.0%
81-46c 3 Act-D825 42.7%/30.2%
81-46c 4 Act-D910 39.2%/28.0%

1. All cells were seeded at a density of 10,000 cells/~l onto amniotic basement membranes in each well.
The data shown are representative of an average of 4 to 6 values per experiment.
2. Cell number was determined by use of a Coulter counter, and percent viability was performed by the Trypan Blue Exclusion Test.
3. Actinomycin D was introduced to the cells on the membrana at a concentration of 0.001~g/ml for the first 24 hours.
According to the present invention, a novel cell culture system may be employed for performing cell invasion assays utilizing a variety' of biological and synthetic membranes. The cell culture system allows utilization of a single membrane for a multiplicity of tests, ~nd allows samples of the cells which have pene-trated the membrane to be taken during the course of the assay.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

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Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A cell culture system for determining cell invasion through an immobilized membrane, said system comprising:
a base plate having a plurality of wells formed in a predetermined pattern therein;
a top plate having a plurality of apertures formed therethrough in a pattern corresponding to that of the wells in the base plate; and means for securing the top plate to the base plate with the apertures and corresponding wells aligned to define test receptacles, so that said receptacles may be partitioned into upper and lower chambers by placing the membrane between the top plate and base plate.

2. A cell culture system as in claim 1, wherein the means for securing the top plate to the base plate comprises a screw.

3. A cell culture system as in claim 1, wherein the wells and the apertures have a circular cross section with a diameter in the range from 0.5 to 2.0cm.

4. A method for performing cell invasion assays, said method employing a culture system comprising:
a base plate having a plurality of wells formed therein in a predetermined pattern; and a top plate having a plurality of apertures formed therethrough in a pattern corresponding to that of the wells in the base plate;
said method comprising:
filling the wells in the bottom plate with a culture medium;

securing the top plate to the base plate with a test membrane therebetween so that the apertures are aligned with the wells and partitioned by the membrane;
seeding cells to be assayed into the apertures above the membrane in a suitable medium;
observing whether the cells are capable of invading the membrane.

5. A method as in claim 4, wherein the test membrane is a human amniotic membrane.

6. A method as in claim 4, wherein the seeded cells are suspected of being neoplastic.

7. A method as in claim 6, wherein the seeded cells are treated with a chemotherapeutic drug prior to seeding.

8. A method as in claim 6, wherein the seeded cells are treated with a chemotherapeutic drug after seeding.