Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Patent
1997-02-12
1999-09-28
Redding, David A.
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
4353051, 4353052, C12M 300
Patent
active
059587628
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a novel device which allows the culture of dissociated cells or organotypic cultures i.e. that of tissular explants on pieces of membrane which have been cut in advance.
STATE OF THE ART
A method described by LStoppini (J. of Neuroscience Method 37 (1991) p. 173) has already been disclosed, in which slices of nervous tissue from rat hippocampus were kept under culture at the interface between the air and a culture medium. The slices, placed on a sterile, transparent and porous membrane (culture insert), were kept in a Petri dish placed in an incubator. Histological and electrophysiological studies made it possible to evidence that this technique allowed explants to be kept alive and that a tissular organisation close to that which can be found in vivo, is retained.
SUMMARY OF THE INVENTION
This invention relates to the use of discs of membrane which have been pre-cut for the organotypic cultures of slices of tissues obtained from the central nervous system.
The disposition of cultures directly on a membrane of an insert has been found during the use, to display some inconveniences which need to be remedied. In fact, in many applications it is then necessary to be in a position to dispose of tissue outside the insert of the culture. It is needed to cut off the membrane around the issue by means of a scalpel.
During this operation, tissues are often damaged. Morever when someone uses the culture inserts, it is not possible to study each implant individually, which has often resulted in a loss of previously cultured tissue.
To remedy these difficulties and to facilitate the handling of organotypic cultures, a novel support has been developed which is constituted of pieces of membrane which are pre-cut and sterile, and which may be circled with a plastic ring.
Thus, the cell cultures are completely localized on the circled discs, so they can develop autonomously and can be removed or replaced at different times.
A more specific subject of the invention is a new device which enables dissociated cells or organotypic cultures, that is to say tissular explants, to be cultured on pieces of membrane cut up in advance.
These pieces of membrane may be uncircled or circled with a ring of rigid or flexible plastic material (eyelet).
Typically, the eyelets are formed by a ring of plastic having a diameter of 3 to 25 mm and a thickness of 0.1 to 1 mm, onto which a permeable culture membrane, commercially-available, such as those sold under the trade names Millicell or Falcon or Millipore or Membrane Anopore (Whatman), is welded or glued. Thus, it is possible to stretch sufficiently the membrane to facilitate the manipulations and treatments necessary in electron microscopy, light-optical microscopy, electrophysiology and for biochemical studies or measurements.
These discs, circled or uncircled, are prepared in advance and are presented in ready-to-use, individual sterile packages.
These discs may be placed: hold several discs, disposed on the median membrane, thus forming what is designated hereafter by the term nursery.
The semi-solid medium has for instance the following composition : (MEM 50%, horse serum 25%, Hank's solution 25%, agarose 0.8-1%). the pH balance is obtained either with bicarbonate and 5% CO2 or HEPES (no need of CO2).
The eyelets are deposited on the surface of the semi-solid medium and thus may be kept for several weeks without any need of changing the culture medium. The eyelets may also be deposited on a removable perforated holder allowing all eyelets to be removed in one operation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a cross section of a device of the invention
FIG. 2 is a cross section of a rigid ring encircling a permeable membrane surface
FIG. 3 is a cross section of FIG. 2
FIG. 4 is an illustration of various membrane provided with a tab for handling uncircled sheets of membrane.
In the device according to this invention, the lower compartment of the nursery contains a membrane which, by capillarity, allows the irrigation of o
REFERENCES:
patent: 4917793 (1990-04-01), Pitt et al.
patent: 4963490 (1990-10-01), Churchouse et al.
patent: 5462874 (1995-10-01), Wolf et al.
patent: 5602028 (1997-02-01), Minchinton
Correges Philippe
Stoppini Luc
Chemodyne S.A.
Redding David A.
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