Methods for the preparation of cell monolayers

Details Methods for the preparation of cell monolayers Methods for the preparation of cell monolayers

1999-03-16

2003-01-28

Le, Long V. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S001100, C435S007100, C435S007210, C435S007920, C435S030000, C435S040500, C435S040510, C435S174000, C435S176000, C435S177000, C435S178000, C435S179000, C435S180000, C435S325000, C435S366000, C435S373000, C435S374000, C435S377000, C435S383000, C435S384000, C435S385000, C435S395000, C435S401000, C435S969000, C436S518000, C436S524000, C436S528000, C436S529000

Reexamination Certificate

active

06511798

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to cell based screens for studying drug transport. The invention also relates to improved methods for the preparation of cell monolayers for use in such screens.
DESCRIPTION OF RELATED ART
Cell based screens have increased in prominence during the last five years, particularly the use of human cell lines for studying drug transport. Cell lines are grown on permeable membrane supports to produce monolayers of cells which are used to examine drug transport via both absorptive and secretary processes. Current tissue culture methodologies for cells are laborious requiring extended culture periods (21 days post seeding onto filters) in order to obtain confluent monolayers of differentiated cells.
Streamlining of the current systems by reducing the time in culture is unsatisfactory since this leads to poor differentiation of the cells and poor expression of certain transporters which are important considerations for drug transport studies. Similarly, new methodology being introduced to reduce the culture time (e.g. Falcon BIOCOAT 3 day system) also results in sub-optimal monolayers in terms of functional expression of certain markers and relatively poor monolayer integrity as shown by the relatively high permeability of “impermeable” markers such as mannitol.
SUMMARY OF THE INVENTION
Our studies have now advantageously shown that it is possible to reduce time in culture dramatically but yet obtain differentiated cell monolayers in seven days or less.
Therefore according to a first aspect of the invention we provide a method for the preparation of a differentiated cell monolayer which methods comprises seeding a permeable charged polymeric support with confluent cells in an appropriate growth medium, incubating the support to allow cell attachment, and replacing the growth medium one or more times, whereby cells grown on the support and form a differentiated cell monolayer.
DETAILED DESCRIPTION OF THE INVENTION
The method of the invention may be used to prepare a cell monolayer of any convenient cell line. This is preferably a human cell line for use in studying drug transport. A particularly convenient cell line is the human adenocarcinoma cell line Caco-2, available from the European Collection of Animal Cell Cultures (ECACC 86010202) CAMR, Porton Down, Sailsbury, Wilshire, UK, SP4 0JG.
The permeable charged polymeric support is preferably collagen coated (to improve cell adhesion) and advantageously transparent. The support is preferably provided in the form of a filter and, for example, shaped to provide apical and basolateral chambers as shown in FIG.
1
.
Therefore in a preferred aspect of the present invention we provide a method for the preparation of a differentiated monolayer of Caco-2 cells which method comprises seeding a permeable charged polymeric support with confluent Caco-2 cells in an appropriate growth medium, incubating the support to allow cell attachment, replacing the growth medium one or more times, whereby the Caco-2 cells grow on the support to form a differentiated Caco-2 cell monolayer.
This system takes a total of seven days with only one, preferably two replacements of the growth medium being necessary to produce satisfactory monolayers. When compared with the routinely used three week system, the process is more cost effective both in terms of reducing the materials used and minimising the degree of manual intervention involved which also helps to reduce the likelihood of contamination.
The growth medium is preferably replaced, in all chambers, about 24 hours after seeding the charged polymeric support for cell growth. This allows any excess cells to be removed. The growth medium is replaced again, about five days after seeding the charged polymeric support for cell growth. By this time the cell monolayer is ready for the preferred addition of a maturation agent as explained hereinafter.
The growth medium comprises any convenient energy and carbon source for the growing cells. A preferred source is L-glutamine, such as a 2 mM solution of L-glutamine, more preferably the more concentrated M1 serum-free supplement (Imperial Laboratories). The growth medium may also comprise one or more of the following: cell nutrients, vitamins, cofactors. Human transferrin is a further ingredient.
Antibiotics, such as penicillin and streptomycin, may be added to the growth medium to inhibit the growth of bacteria.
Monolayers produced have acceptable barrier properties for assessing passive permeability of drugs (based on restricted permeability of mannitol), and show functional expression of the dipeptide carrier and p-glycoprotein. The ability to produce viable Caco-2 cell monolayers within seven days also provides greater scope for applying the system early in drug candidate selection.
The permeable charged polymeric support is conveniently polyethylene terephthalate (PET), polycarbonate, cellulose acetate, cellulose nitrate, mixed polymer, preferably polyethylene terephthalate (PET). Whilst we do not wish to be bound by theoretical considerations, it is believed that a PET support allows better initial cell attachment and subsequent growth.
The M1 serum-free supplement used in this method contains a mixture of amino acids, vitamins, trace elements, other organic supplements and transferrin. It helps maintain viability of high density cultures when used in conjunction with media containing serum by providing a nutrient-rich supplement in a convenient form.
A further advantage is that a maturation agent can be used to good effect in the method of the invention. This helps to prevent leakiness between cell junctions and promotes an intact cell monolayer. In general the maturation agent is added about 5 days after seeding. Any convenient maturation agent may be used, preferred agents include butyric acid and sodium butyrate.
Not only does the method of the invention provide a more efficient process for preparation of integral confluent monolayers but also these monolayers have significantly improved membrane barrier properties (reduced mannitol permeability) and good expression of biochemical markers (alkaline phosphatase) compared to existing protocols.
By “a differentiated cell monolayer” we mean a cell monolayer that expresses a mature phenotype and is suitable for use in screens to study drug transport. In the case of Caco-2 cells, this may be characterised by one or more of the following features: a high density of microvilli on the apical surface, desmosomes, tight junctions as measured by permeability to mannitol (P
app
equivalent to <8×10
−7
cm/s and generally around 4×10
−7
cm/s), expression of a p-glycoprotein dipeptide transporter and reliable expression of differentiation markers such as alkaline phosphatase.
In a further aspect of the invention we provide differentiated cell monolayers prepared according to the methods of the invention.
A further significant advantage is that the same or different cell monolayers may be grown on different surfaces of the charged polymeric support. Conveniently the cell monolayers may be grown on opposite surfaces for example opposite sides of the charged polymeric support. (e.g. Astrocytes/endothelial cells: endothelial cells
euronal cells: hepatocytes/tumour epithelial cells etc.) In a preferred embodiment, two different cell monolayers are grown on opposite sides of a charged polymeric support to form a bilayer culture, for example as shown in
FIG. 2
a
. In a further preferred embodiment, two different cell monolayers are grown on opposite sides of the charged polymeric support and a third target cell monolayer is grown in the culture dish to form a bilayer culture with target cell, for example as shown in
FIG. 2
b
. Therefore in a further aspect of the invention we provide a permeable charged polymeric support having differentiated cell monolayers (which may be the same or different) on different surfaces of the support. It will be appreciated that the support may be provided in a number of shapes and will be limited only by practical co

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