WO1990015862A1

WO1990015862A1 - In vitro cultivation of epithelial cells

Info

Publication number: WO1990015862A1
Application number: PCT/US1990/003305
Authority: WO
Inventors: Fred Quimby; Raoul F. Reiser
Original assignee: Cornell Research Foundation, Inc.
Priority date: 1989-06-12
Filing date: 1990-06-11
Publication date: 1990-12-27

Abstract

A method is described for the in vitro cultivation of epithelial cells. Utilizing a population of fibroblast cells separated physically from the epithelial cells as a feeder layer (21) for the epithelial cells, differentiation of the epithelial cell layer (41) is obtained.

Description

IN VITRO CULTIVATION OF EPITHELIAL CELLS

In 1979, toxic shock syndrome (TSS) was recognized as a menstrually related disease, and was quickly demonstrated [see Bergdoll et al, A new staphylococcal enterotoxin, enterotoxin F, associated with toxic shock syndrome Staphylococcus aureus isolates, Lancet 1 :1017 (1981)] to be caused by a toxin produced in the vagina by Staphylococcus aureus. Recently it has been shown that large concentrations of this toxin could be present in the vagina of healthy nonhuman primates with no detectable amounts found in the blood stream [see Quimby et al, Preliminary observations on the ability of tampons to support the growth of Staphylococcus aureus and toxic shock syndrome toxin-1 secretion during menstruation in baboons, Rev. Infect. Dis. Suppl. 11 :S332 (1989)]. Based upon these and other reports in the literature, it has been postulated that the human vaginal mucosa normally provides a barrier to the transport of TSS toxin into the bloodstream, and that this barrier is somehow overcome by the use of tampons. In order to understand the workings of the vaginal mucosal barrier, it is essential to know which factors regulate transepithelial toxin transport and how these factors are modified in patients afflicted with TSS. The transport of macromolecules across keratinizing and nonkeratinizing epithelial layers in humans and nonhuman primates has not been extensively studied. While transport across oral mucosa, skin and intestinal mucosa has been studied, only cursory attention has been paid to the vaginal mucosa, the last publication having been made in 1983 (see King, The permeability of non-human primate vaginal epithelium: a freeze-fracture and tracer-perfusion study, J. Ultrastruct. Res. 83:99). Major drawbacks preventing these studies in humans and nonhuman primates are cost, time, hormonal regulation within individual animals, and availability. Even when cost may be justified, the availability may be the limiting factor.

Since initial experiments on animal models were precluded, in vitro testing systems were studied. While there are a number of tissue culture systems that permit the cultivation and study of non-typical human epithelial cells in vitro, there are few reliable culture systems that permit the study of normal human epithelial cells. Among the factors contributing to the relatively low success rate in achieving epithelial cell tissue cultures [see Owens et al, Epithelial cell cultures from normal and cancerous human tissues, J. Natl. Cancer Institute 56:843 (1976)] is a slow doubling time as compared to fibroblasts, explant contamination with fibroblasts, and lack of essential nutritional requirements from the growth media.

With these factors in mind, the present invention was developed initially as an in vitro test system to test the adsorption of toxic shock syndrome toxin-1 (TSST-1) across vaginal mucosae of both human and nonhuman primate origin (in addition to this specialized use, the present invention may also be used for other purposes requiring the involvement of other mucosal epithelial cells - cervical, conjunctival, mammary canal, buccal, nasal, etc. - as for example, the transportation of molecules across cellular surfaces).

Specifically, the present invention provides a method for obtaining a raised in vitro tissue culture which employs a multi- layered, differentiated, epithelial cell layer supported on a collagen membrane with multi-layers of fibroblasts on the undersurface of the membrane. Although this is not the first description of an epithelial tissue culture [(see Sobel et al, Human vaginal epithelial multilayer tissue culture, In Vitro, 15(12):202 (1979)], the present invention using a raised differentiated epithelium overlaying a "stroma" of collagen and fibroblasts approximates the in vitro situation to a higher degree than does a system growing directly on plastic. In addition, the present invention allows the study of mucosal surfaces from beneath the "stroma" as well as from the exposed exterior side of the mucosal surface.

This and other aspects of the present invention will become apparent from the following detailed description of one preferred embodiment for obtaining a multi-layered, differentiated, vaginal squamous epithelial cell layer which is to be taken in conjunction with the accompanying drawings in which like numerals indicate like components and in which:

FIG. 1 is a cross sectional plan view of a covered circular tissue culture dish containing a membrane support; FIG. 2 is a cross sectional plan view of the culture dish depicted in Fig. 1 showing cellular seeding of the fibroblast cell layer;

FIG. 3 is a cross sectional plan view of the culture dish depicted in Fig. 1 showing cellular seeding of the epithelial cell layer;

FIG. 4 is a cross sectional plan view of the culture dish depicted in Fig. 1 showing a multi-layered, differentiated epithelial cell layer obtained according to the method of the present invention. With specific reference to Figure 1 , a circular tissue culture dish 10 is shown as having bowl 12 and a cover 11 portions and a removable membrane support 13 which comprises a circumferential ring support 14 and a membrane 15 within the ring support. Such tissue culture dish and supports are commercially available from ICN Biomedicals, Inc. (Cleveland, Ohio) as part of their total CellagenTM tissue culture system. The membranes of this system are permeable, pepsin-solubilized collagen films developed for tissue culture use which permit the characteristic permeation of substances (such as the absorption of nutrients and passage of metabolites) through the membrane. Although other raised culture systems are available, in order to achieve the required growth of cells to confluence, the material of the support 14 for the membrane film 15 must be compatible with the cells being grown - polycarbonate or glass are preferred. When systems using polystyrene supports were used for example, the polystyrene was found to inhibit cell growth to the extent that while growth occurred, it would not extend completely across the face of the membrane.

In conventional usage, a sufficient amount of culture or nutrient media 22 is poured into the bowl 12 of the culture dish 10 to cover the membrane support 13 when it is next placed into the dish. A suspension of cells 21 is then placed upon the upper surface of the membrane 15, additional media is added if needed to cover the seeded cells, the dish covered, and the dish placed into an appropriate tissue culture incubator and the cells allowed to grow.

In addition to providing for cell culture on one side of the cellagen membrane, cells may be cultured on both sides of the cellagen membrane. According to the conventional protocol for such two-sided culture, cells are first placed upon the upper surface of the membrane 15 and the cells 21 are cultured as usual. After confirming that the cells have adhered to the membrane, the support is removed, reversed so that the adhered cells will be on the lower surface of the membrane 15, replaced into the dish once again, and the upper surface 31 of the membrane is seeded with a second type of cell 32 and culture media is added to ensure that the seeded cells are below the surface of the media. Following a second incubation, the membrane 15 will have one cell type on its upper surface, and a second cell type on its lower surface.

These general protocols for use of the CellagenTM membrane tissue culture system require certain specific modifications in order to function in the present invention. The first of these modifications is that the initial layer of cells needs to be fibroblasts which will produce the necessary exogenous hormonal and nutritional requirements required for epithelial cell differentiation and layering; the second modification is that the initial cell layer must have grown to confluence (rather than merely adherence) across membrane 15 prior to reversing the support 13 in the bowl; and the third modification is that the epithelial cells require a cellular-air interface 33 in order to differentiate - simply put, the initial cellular layer must be bathed in the nutrient media in order to allow cellular growth (Fig. 1), however, the nutrient solution must not cover the upper surface of the epithelial layer of cells if they are to differentiate. A third, but optional, modification is that the size of the culture dish 10 should be greater in relation to the size of the support 13 than presently provided in the commercial tissue culture kits. Utilizing this modified protocol, up to fifteen layers of differentiated mucosal epithelial cells have been prepared in tissue culture.

These and other modifications to conventional tissue culture techniques will become apparent to from the following example.

EXAMPLE 1

Full thickness vaginal biopsy specimens were obtained aseptically from adult female baboons maintained under general anesthesia. The biopsy specimen was placed in 15 to 20 ml of Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10 times the normal concentration of Penicillin-Sreptomycin- Amphotericin B (Antibiotic-Antimycotic) (Ab/Am) for a minimum of two hours. The specimens were then transferred, epithelial surface down, to a petri dish containing DMEM-1x Ab/Am and the connective tissue was carefully removed using curved surgical scissors. The tissue was then processed according to a modified procedure of Wilkinson et al [J. Invest. Dermatol. 88(2) :202]. The tissue was minced using the crossed scalpel technique. Individual pieces of tissue were dipped into chick embryo extract (available from GIBCO, Grand Island, New York), and placed individually into walls of twelve well tissue culture plates. The explants were then allowed to adhere to the plastic by drying at room temperature in the laminar flow hood for no more than 10 minutes. At this time, one drop of chicken plasma (available from Cocalico Biologicals, Reamstown, Pennsylvania) was placed over each explant. One-half to one drop of sterile 0.025 M calcium chloride solution was placed on each plasma drop to facilitate clotting (approximately 20 to 30 minutes). Next, the plasma clot containing explant was surrounded with William's Modified E Medium (available from GIBCO) containing 10% fetal bovine serum (available from Intergen Co., Purchase, New York), 0.5% Dimethyl Sulfoxide (DMSO) (available from Sigma Chemical, St. Louis, Missouri), 10 ng/ml Epidermal Growth Factor (EGF) (available from Collaborative Research Laboratories, Lexington,

Massachusetts), 10"^"- °M Cholera Toxin (CT) (available from ICN Biomedicals, Costa Mesa, California) and Ab/Am. After adding the DMSO-medium, the explants were placed in a 37°C - 5% Cθ2 tissue culture incubator. After overnight incubation of the explants in DMSO-medium, the volume of medium was tripled (0.5 ml to 1.5 ml per well in a 12 well plate) using the same medium formulation without DMSO (Complete Medium). Epithelial cells began to emerge from the explant between 4 and 10 days. Pieces of connective tissue trimmed from the biopsy specimen were treated in a similar fashion in order to obtain fibroblasts which were to be used for "feeder layers".

Usually within two weeks the wells were ready for the first harvest of epithelial cells. Using a gentle harvest technique (0.25% trypsin in calcium-magnesium free EDTA-HEPES buffer), the cells were harvested and transferred either directly to Cellagen membranes or expanded using 25 cm² tissue culture Nunc flasks. With careful trypsinization and handling, the original explants continued to yield epithelial cells for as long as three months. Cellagen membrane systems (33 mm in diameter) were set up in the following manner. Seven ml of complete medium was added to the 60 mm dish containing the membrane as supplied. Considerable care must be taken in order to avoid and/or remove air bubbles trapped under the membrane. Two ml of buffer containing 1 x 1θ4 fibroblasts per ml were then added to the empty cellagen membrane chamber in such a manner that uniform distribution of the cells was achieved. The membrane system, containing the fibroblasts, was then incubated until the fibroblasts were confluent across the face of the membrane. At this time, the Cellagen disc with the fibroblast monolayer on the top side was inverted using a sterile forceps, and aseptically transferred to a 100 mm petri dish containing 17 ml of complete medium in such a way that the medium made uniform contact with the fibroblast monolayer. Epithelial cells from the previously prepared explants were then seeded on top of the cellagen system (2 ml of medium containing 1 x 10$ cells/ml). The entire system, i.e. the 100 mm petri dish containing 17 ml of complete medium in the dish, the cellagen membrane with fibroblasts underneath and 2 ml of medium containing epithelial cells within the surface well of the membrane disc, was then incubated for 48 hours before changing medium.

After 48 hours, the medium was changed by removing approximately 12 ml of medium from the petri dish using precautions to avoid drawing air under the disc. The medium on the upper surface of the disc was removed gently so as not to tear or disturb the surface of the membrane. Twelve ml of fresh medium was then returned to the lower chamber, and one ml was replaced on the top of the membrane to maintain the cells within a moist environment (the upper surface of the cells were maintained in contact with the moist air in the covered tissue culture dish). The system was then returned to the 37°C - 5% CO2 incubator until the next medium change.

The medium was changed 2 to 3 times per week depending upon the phenol red indicator. In three to five weeks of incubation, the epithelial cells were differentiated and in layers of five to nine cell layers thick.

Utilizing minor modifications of the method described in Example 1 and the apparatus depicted in the figures, it has been possible to show that cultures can be controlled so that they yield essentially only epithelial cells. Photomicrographs of such tissue culture clearly demonstrated that these epithelial cells differentiate in a normal fashion as evidenced by the presence of desmosomes and interlacing microvilli. It has also been seen that the fibroblasts may also differentiate in this system into multiple layers of metabolically active cells which, together with the collagen membrane, for ma "stroma" beneath the epithelial cells. Diffusable substances can traverse this stroma which allows for the epithelial cellular layers to receive nutrition from the stromal layers without directly becoming in contact with the fibroblast cellular layer. Utilizing the culture system according to the present invention, the epithelial cellular layer was found to contain the morphologic and ultramicroscopic features of epithelial cell differentiation necessary to study the absorption and transport of TSST-1. Likewise, the influence which host derived and non host derived substances may have on toxin transport across the vaginal epithelium may be assessed. These future studies should provide an insight into how TSST-1 crosses the vaginal membrane and thus initiates the sequence of events resulting in toxic shock syndrome. Furthermore, studies using various epithelial cell systems should provide insight into the mechanisms for drug and nutrient absorption as well as toxin transport. This system also allows for the evaluation of compounds which may act through fibroblasts to exert their ultimate effect on epithelial cell growth and differentiation. By probing the system with microbial agents, both invasive and noninvasive, information relevant to receptor and non receptor mediated adherence, epithelial cell infection, and intracellular proliferation of microorganisms may be obtained. Likewise those host factors indigenous to epithelial cells which are important for the prevention of infections may also be studied.

Thus, while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification, and we therefore do not wish or intend to be limited to the precise terms set forth, but desire and intend to avail ourselves of such changes and modifications which may be made for adapting the present invention to various usages and conditions. Accordingly, such changes and modifications are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims. The terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not of limitation, and thus there is no intention, in the use of such terms and expressions, of excluding equivalents of the features shown and described, or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.

Having thus described our invention and the manner an process of making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or to with which it is most nearly connected, to make and use the same,

Claims

WE CLAIM:

1. A method for the in vitro growth of mucosal epithelial cells which comprises: a. providing a membranous support film upon which to grow mucosal epithelial cells; b. seeding one side of said film with a cell suspension of fibroblast cells; c. allowing said suspension to grow within a nutrient medium to confluence across the face of said film; d. seeding an opposite side of said film with a cell suspension of mucosal epithelial cells; and e. allowing said epithelial cell suspension to grow and layer upon said film.

2. A method according to Claim 1 which further comprises providing the epithelial cells in the suspension with a cell-film interface and a cell-air interface.

3. A method according to Claim 2 which further comprises allowing the epithelial cells in suspension to grow to confluence and to form multiple layers over a period of time.

4. A method according to Claim 1 wherein the epithelial cells are selected from the group consisting of vaginal, buccal, mammary canal, nasal and conjunctival epithelial cells

5. A tissue culture system comprising: a. a dish for holding a nutrient tissue growth medium; b. a removable cover for said dish; c. means within said dish for providing a permeable membranous film wherein said means has an upper surface facing said cover and a lower surface facing said dish; d. fibroblast cells adhered to the lower surface of said means, and mucosal epithelial cells adhered to the upper surface of said means.