U.S. patent application number 10/252804 was filed with the patent office on 2003-01-30 for arrangement for treating cells in biology or genetic engineering.
Invention is credited to Marotzki, Stefan.
Application Number | 20030022365 10/252804 |
Document ID | / |
Family ID | 8063135 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022365 |
Kind Code |
A1 |
Marotzki, Stefan |
January 30, 2003 |
Arrangement for treating cells in biology or genetic
engineering
Abstract
A device for receiving a liquid containing a cell culture
includes at least one vessel having a base plate and walls forming
an enclosure. A cover is lowerable into the vessel to displace air
and, where appropriate, excess liquid. The cover has a liquid
admission opening and a liquid discharge opening.
Inventors: |
Marotzki, Stefan; (Tornesch,
DE) |
Correspondence
Address: |
ALIX YALE & RISTAS LLP
750 MAIN STREET
SUITE 1400
HARTFORD
CT
06103
US
|
Family ID: |
8063135 |
Appl. No.: |
10/252804 |
Filed: |
September 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10252804 |
Sep 23, 2002 |
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09787948 |
Mar 23, 2001 |
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6468788 |
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Current U.S.
Class: |
435/305.4 ;
435/305.3 |
Current CPC
Class: |
C12M 23/46 20130101;
C12M 23/10 20130101; C12M 23/38 20130101; C12M 25/02 20130101 |
Class at
Publication: |
435/305.4 ;
435/305.3 |
International
Class: |
C12M 001/22 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 1998 |
DE |
298 17 223.2 |
Oct 27, 1998 |
EP |
98 120 337.5 |
Sep 13, 1999 |
WO |
PCT/EP99/06762 |
Claims
What is claimed is:
1. A device for receiving a liquid containing a cell culture
comprising: a vessel having a base plate and a wall structure
defining an enclosure; and a cover lowerable into the vessel to
displace air and, where appropriate, excess liquid, said cover
having a liquid admission opening and a liquid discharge
opening.
2. The device according to claim 1 further comprising one or more
walls for defining a plurality of separate chambers inside the
vessel.
3. The device according to claim 2, wherein said vessel has a first
width and further comprising a second vessel having a second width
which is less than said first width, said second vessel being
disposed in said first vessel.
4. The device according to claim 3, wherein said cover closes only
the portion of the wider first vessel which is situated outside of
the second vessel.
5. The device according to claim 3, wherein the cover defines a
cutout which is configured to be complementary with the
circumference of the second vessel.
6. The device according to claim 5, wherein the cutout is defined
by an opening of the cover.
7. The device according to claim 3, wherein the cover is a holder
for the second vessel.
8. The device according to claim 7, wherein the cover connects with
the second vessel to permit joint handling.
9. The device according to claim 3 further comprising a separate
cover for covering the second vessel.
10. The device according to claim 2 further comprising a ring
enclosing a separate chamber disposed between the base plate and a
base of the cover and the cover has an opening communicating with
the chamber.
11. The device according to claim 1, further comprising at least
one cell culture insert.
12. The device according to claim 1, wherein at least one of the
openings is provided with a screen.
13. The device according to claim 1, wherein the cover has an upper
side defining a trough in fluid communication with at least one of
the openings.
14. The device according to claim 13, wherein the trough has a
lower base which is not disposed lower than a mouth of the
associated opening.
15. The device according to claim 14, wherein the upper side of the
cover defines first and second troughs associated with the liquid
admission opening and liquid discharge opening, respectively, the
first trough being separated from the second trough.
16. The device according to claim 13, wherein a plurality of
vessels are enclosed by the cover, the trough being in fluid
communication with each of the vessels through a one of the
openings disposed proximate to the vessel.
17. The device according to claim 1, wherein the cover has a
plurality of openings, at least one of the openings being a liquid
admission opening and at least one of the openings being a liquid
discharge opening.
18. The device according to claim 1, wherein the device comprises a
plurality of contiguous vessels.
19. The device according to claim 4, wherein the cover defines a
cutout which is configured to be complementary with the
circumference of the second vessel.
20. The device according to claim 4, further comprising a separate
cover for covering the second vessel.
21. An arrangement for biological treatment or genetic engineering
of cells comprising: at least one vessel each having a base plate
and a wall defining an enclosure for receiving cells and a
treatment liquid; a machine having means for exchanging the liquid;
and a cover having at least first and second openings; wherein the
liquid exchange means introduces liquid into the first opening and
removes it from the second opening.
22. The arrangement of claim 21, wherein said cover has an upper
side defining first and second troughs in fluid communication with
the first and second openings, respectively, and the liquid
exchange means introduces liquid into the first trough and removes
it from the second trough.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/787,948, filed on Mar. 23, 2001.
BACKGROUND OF THE INVENTION
[0002] For treating or examining cells in molecular biology or
genetic engineering, vessels are used (for example, Petri dishes)
which receive the cells and a reaction liquid and can be provided
with a cover ("Lab-Tek II" brochure from Nalge Nunc International,
Naperville;
[0003] "EasiSeal" brochure from HYBAID Limited, Teddigton; "Gene
Frame" brochure from Advanced Biotechnologies Ltd., Espom; EP-A 611
598; U.S. Pat. Nos. 4,634,676; 3,745,091; DE-A 19624 917; DE-C 479
304; U.S. Pat. Nos. 2,348,448; 4,294,924; 4,321,330; FR-A 2 698
375). If the treatment (for example, in situ hybridization or in
situ PCR) requires exchange of liquids, this can only be done
manually in most of the known vessels, because it involves removal
and application of a cover. If the treatment requires rapid
temperature change, it must be ensured that the substrate layer in
the vessel is very thin so as to be able to follow the temperature
change quickly and in a precisely controllable manner. In
treatments in automatic machines, these requirements have hitherto
been satisfied using vessels (Lab-Tek II brochure from Nalge Nunc
International, Naperville; U.S. Pat. No. 3,745,091) which consist
of a slide with walls detachably arranged thereon. In a first
method step which takes place outside the automatic device, the
vessels are used with the walls placed on the slides, in which case
liquid exchange does not take place or has to be carried out
manually. As soon as the automatic treatment is to start, the walls
are removed and the slides with the cells adhering to them are
introduced into the automatic machine in which the liquid exchange
takes place by means of the slides being lowered in groups into
optionally alternating baths. If the cells do not adhere, or adhere
insufficiently, to the slide, and they are instead completely or
partially in suspension, they are centrifuged on the slide so that
they adhere thereto after removal of the medium. This is not only
complex but also has the disadvantage that the cells are deformed
and slight morphological changes can no longer be identified under
the microscope. In addition, the required centrifugation steps can
not be automated or can be automated only with difficulty.
[0004] The removal of the walls from the slides complicates the
procedure, particularly in the case of cells adhering inadequately
to the slide. Finally, it may be undesirable to eliminate the
vessel division between adjacent cultures by removing the
walls.
[0005] It is admittedly known (FR-A-2 698 375) to provide a Petri
dish with a cover which has an opening. This is used for removing
air and excess liquid when the cover is lowered into the vessel.
However, this does not include the concept of automatic liquid
exchange using the cover opening as an admission opening and
outflow opening.
SUMMARY OF THE INVENTION
[0006] It is an object of the invention to make available a device
for receiving a liquid containing a cell culture comprising a
vessel having a base plate and walls defining an enclosure, and a
cover lowerable into the vessel to displace air and, where
appropriate, excess liquid, the cover having a liquid admission
opening and a liquid discharge opening.
[0007] The cover can further comprise a plurality of openings,
where at least one opening is a liquid admission opening and at
least one opening is a liquid discharge opening.
[0008] If displaced liquid is to be collected, or if a reservoir of
liquid is to be formed which is intended to replace the liquid
present in the treatment chamber, a trough can be formed on the
upper side and communicate with a corresponding opening in the
cover.
[0009] For liquid which is to pass into the treatment chamber by
diffusion or flow, the trough base should not be lower than the
mouth of the opening. A plurality of openings can be provided per
vessel in order to permit liquid exchange by flow. For this
purpose, a plurality of openings should be provided, of which at
least one is connected to a trough which is separate from the
trough or troughs of the other openings and which is not arranged
lower than the mouth of the one opening.
[0010] Particularly in machine treatment of the devices, it may be
expedient to combine a plurality of vessels and provide them with a
common cover which has the requisite openings in the area of each
individual vessel.
[0011] The troughs have the advantage that the mechanical means for
introducing liquid can be designed alternately for introduction
into the openings or into the associated troughs. Similarly, they
can be alternately designed for removing liquid off from a trough
or opening.
[0012] In this context, the term trough is intended to signify any
vessel-like depression without stating its width or height, unless
this is expressly mentioned.
[0013] The cover can, as is known, be designed such that air and a
possible excess amount of reaction liquid is displaced through the
opening. The cover can be lowered inside the vessel until the
desired thickness of the substrate layer is achieved between the
base plate of the vessel and the opposite cover base, which desired
thickness can range from several hundredths of a millimeter to
several millimeters.
[0014] In order to avoid undesired void volume, the circumferential
surface of the cover can fit closely, with an essentially identical
shape, to the internal surface of the walls. This fit can also
constitute the sealing of the cover with respect to the walls, for
example, by means of a ground-glass joint or a plastic seal under
elastic pressure. However, this does not need to be the case,
since, in addition to a close, but not tight fit of the
circumferential surface of the cover to the internal surface of the
walls, a special seal can be provided which can cooperate, for
example, with the edge of the vessel walls or a shoulder thereof.
In these cases, it is often expedient to provide a holder for
maintaining the sealing position of the cover with respect to the
vessel, for example, a screw-on, snap or spring closure; however,
the tight fit of the cover on the vessel walls can also be
self-supporting, for example, as a result of the frictional forces
which exist between two ground-glass surfaces.
[0015] It is expedient for the lower surface of the cover base to
extend approximately parallel to the base of the vessel in order to
be able to achieve an essentially constant layer thickness. The
cover and the vessel can be provided with cooperating contact
surfaces, which define the size and constancy of the layer
thickness.
[0016] The openings of the cover can be provided with a closure
device. However, the possibility also exists of providing sealing
by means of a subsequently applied oil layer. This also applies to
the circumferential sealing. In many cases a tight seal is only
required in respect of the external atmosphere in order, for
example, to avoid evaporation or the admission of oxygen. In these
cases, it may suffice if the seal is formed not on the vessel and
cover, but is instead formed by an apparatus which receives the
device, for example, a type of autoclave in which there is an
internal atmosphere which is chosen in accordance with the intended
purposes. To avoid evaporation, for example, it can have sufficient
moisture. To avoid admission of harmful gases, it can consist of
nitrogen or noble gas.
[0017] To ensure that it is not only the air in the area of the
cover opening that is removed, but also that air quantity which may
be contained between the circumferential surface of the cover and
the vessel walls, it is possible for the cover opening to be
provided with an ascending section whose mouth is at least
approximately level with the upper end of the gap situated between
the circumferential surface of the cover and the vessel walls. On
slow insertion of the cover into the vessel, the static pressure of
the liquid column present in the ascending section of the cover
opening then ensures that the air in the circumferential gap is
also displaced.
[0018] When the cells have deposited sufficiently firmly on the
base of the vessel, the excess liquid can be drawn off or displaced
through the cover opening without the risk of losing cells. So that
it is also possible to work with cells, which are in suspension,
the cover opening can be provided with a screen which holds back
the cells during displacement of the liquid.
[0019] A plurality of openings can be provided which are designed
for attachment of an admission and discharge line for a medium. The
device can then also be used as a so-called reactor (Meenen et al.;
"Semi Continuous Reactor System . . . ", Poster 1994 Biomaterials
21:905-908).
[0020] The cells involved can be adherent cells, which stick to the
base of the cell culture dish, or suspension cells which swim in
the culture medium. Alternatively, tissue sections can also be
cultured in the dish. In general, both eukaryotic and prokaryotic
cells can be cultured in the vessel. Treatments and examinations
which can be carried out using the device according to the
invention can, for example, be all types of in situ hybridization
and in situ PCR.
[0021] The cover openings are expediently provided inside the
cover, at a distance from the edge of the latter. However, this
does not exclude the possibility of an opening being formed by
means of a spacing provided, at least in places, between the edge
of the cover and the vessel walls. A particularly advantageous
design in this connection is one in which the edge of the cover
comprises a collar which rises upwards from the bottom of the cover
plate, which forms in its entirety or in places the said spacing
for forming an opening, and whose upper edge cooperates with the
vessel walls to form a seal. As the cover is being lowered,
quantities of gas and liquid enclosed between cover and vessel can
then escape, and it is only at the end of this procedure, when the
cover reaches its end position that the seal is obtained.
[0022] If the intention is to subject a plurality of cultures to
the same thermal conditions, it is expedient to connect a plurality
of vessels to one another. In this case, a cover of the type
indicated above, can be provided for each one of the vessels.
However, it is also possible to connect a plurality of covers to
one another for joint actuation. It is of course possible to make
the walls of a plurality of vessels integral with one another, in
which case either the bases are also connected in one piece with
the walls or they can be separated from these, for example in the
form of a slide.
[0023] Of particular importance in the context of the invention is
the possibility of being able to create a plurality of chambers by
means of forming walls inside one and the same vessel, which
chambers, during the course of treatment of a culture, are at times
separated from one another, or are not separated, depending on the
requirements. For this purpose, the cover is provided with an
arrangement for dividing off such chambers from one another. A
particularly advantageous design is one in which a narrower vessel
is so designed that it can be accommodated in a wider vessel. The
cover of the wider vessel is in this case generally designed in
such a way that it serves solely to close the area of the wider
vessel situated outside the narrower vessel. For the closure of the
narrower vessel, a special closure is then optionally provided, for
which the general comments made above in respect of the closure of
the vessels also apply. This means that in addition to a simple
air-permeable or airtight closure, a cover can also be provided
which is equipped with an opening for displacement of the
atmosphere, and possibly excess liquid, arising in the narrower
vessel, and whose air opening can be closed. However, there is also
the possibility of structurally connecting the covers of the wider
vessel and of the narrower vessel to one another for joint
actuation.
[0024] Where, in the present connection, mention is made off "a"
narrower vessel, this is intended to mean that it is at least one;
a plurality of narrower vessels can, however, also be provided in a
wider vessel. The arrangement of a narrower vessel in a wider
vessel affords the possibility of subjecting the cultures within
the narrower vessel and within the wider vessel to exactly the same
thermal conditions. They can have separate bases. Of particular
importance, however, is a design in which the narrower vessel
shares the base with the wider vessel. This affords the possibility
of subjecting one and the same culture inside the wider vessel to
different reaction conditions (for example, another reaction
liquid) during all or some of the reaction steps. Accordingly, the
wall of the narrower vessel is used only in those reaction steps,
inside the otherwise uniform culture, in which the reaction
conditions are intended to be different, e.g., for positive control
or negative control in PCR.
[0025] There are cases in which the narrower vessel can remain in
the wider vessel during the entire reaction or sequence of
reactions. In these cases, it can be permanently connected to the
base and to the wider vessel from the outset at the factory stage.
In other cases, the compartmentalization is desired at the start of
the reaction or chain of reactions; the narrower vessel can then be
connected, likewise at the factory stage, to the base of the wider
vessel, but it can be detached therefrom so that the
compartmentalization can be annulled at the desired time. The
factory-stage arrangement has the advantage that the narrower
vessel can be accurately positioned in relation to the walls of the
wider one, and this therefore guarantees that the cover fits. In
other cases, one will want to have the freedom to be able to carry
out the compartmentalization at any desired time. In these cases,
the wall of the narrower vessel can be fitted by the user.
[0026] If the cover is intended to close off the wider vessel
completely, it must not only have its circumferential surface
adapted to the wall of the wider vessel, but must also be able to
be connected sufficiently tightly to the narrower vessel. This can
be achieved by its having a cutout which is adapted to the shape of
the narrower vessel and which can join to the latter with the
desired tightness. This cutout can be formed by an outflow opening.
In another embodiment, the cover is connected in one piece to the
wall of the narrower vessel, so that the compartmentalization made
possible by the narrower vessel comes about with the insertion of
the cover. In both cases, the cover serves as a holder or guide
during insertion and, if appropriate, also during use of the
narrower vessel.
[0027] In some cases an absolutely tight seal between the wall of
the narrower vessel and the base of the wider vessel is not
necessary; the narrower vessel can then simply be formed by a small
tube which is held by the cover and reaches down to the base of the
wider vessel. If a tight connection of the narrower vessel wall
against the base is required, the lower edge of the wall of the
narrower vessel can be provided with a seal, the wall being pressed
against the base by the cover or an additional clamp in order to
generate the sealing pressure. The wall of the narrower vessel can
also be bonded to the base. In the simplest case, the vessel wall
of the narrower vessel comprises a sealing ring which is fitted
between the base plate and the cover base, fits tightly against
these and divides the chamber enclosed by it from the space
surrounding it. To ensure that the enclosed chamber can be
subjected to a different treatment, it must be accessible by way of
a cover opening which is located in its area and which can be the
outflow opening.
[0028] According to the invention, the cover can also be provided
with further arrangements, which are desired, for the treatment of
the cultures. For electroporation, the cover and the base can
support electrodes, for example, planar electrodes, and the cells
to be treated are arranged on a porous membrane between these.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The invention is explained in greater detail below with
reference to the drawing, which diagrammatically depicts
advantageous illustrative embodiments, and in which:
[0030] FIG. 1 shows a schematic representation;
[0031] FIG. 2 shows a device with ground-in cover;
[0032] FIG. 3 shows a device with elastically sealing cover;
[0033] FIG. 4 shows a device with a screw-on cover;
[0034] FIG. 5 shows a device with a snap-locking cover;
[0035] FIGS. 6, 7, and 8 show different embodiments of a
compartmentalized device;
[0036] FIG. 9 shows a partial section illustrating one sealing
possibility;
[0037] FIG. 10 shows a device with electrode;
[0038] FIG. 11 shows a further compartmentalized device;
[0039] FIG. 12 shows a device with a plurality of vessels and
common cover;
[0040] FIG. 13 shows the device according to FIG. 12 with special
recesses at the cover openings;
[0041] FIG. 14 shows the device according to FIG. 12 with a
plurality of openings per vessel; and
[0042] FIGS. 15 and 16 show a vessel with a plurality of openings
in various stages of operating.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The device basically comprises the vessel 1, for example, a
Petri dish, and a cover 2. The vessel 1 has a base plate 3 and
walls 4. These can be designed in one piece or as multiple parts.
FIG. 3 indicates that the base plate is formed by a slide 3 feet on
which the walls 4 are secured in a known manner by means of a
releasable adhesive 5. Instead of this, a sealing ring can be used
in conjunction with an arrangement for holding the walls, with
inclusion of the sealing ring on the slide. Vessel and cover are
made of a material, which does not negatively affect the substrate,
and the process that may be carried out, for example, glass or
suitable plastic. The vessels may be individual vessels;
alternatively, a plurality of vessels are provided contiguously as
one group.
[0044] If the intention is to cultivate the cells in the dish or to
subject them to molecular biological or genetic engineering
treatments or examinations, the cover 2 is used, which cover 2
closes the dish off tightly against the walls 4. It has at least
one opening 6. The atmosphere which is displaced upon insertion of
the cover into the vessel and, where appropriate, a part of the
liquid, can flow off through this opening. The lower mouth of the
opening is arranged in the part of the cover base 7 submerging in
the liquid, and in fact it is preferably arranged in the highest
region thereof. If so required, the opening can be closed after the
cover has been applied. In this respect, the vessel is known (FR-A
2698375). The distance between the bottom surface of the cover base
7 and the top 8 of the base generally amounts to 20 .mu.m for
observation purposes, but can also be substantially larger or
smaller, depending on requirements.
[0045] The type of sealing of the cover 2 with respect to the walls
4 which is chosen in each particular case depends on the material
that has been chosen. If glass is used, this can be achieved by
means of grinding. This is illustrated in FIG. 2. The cover 2 and
the vessel 1 are provided with collars, 9, 10 which have been
ground to match and complement each other. The cover opening 6 is
situated at a suitable location within the cover area surrounded by
the collar. Such a shape of the vessel and of the cover can also be
used with materials other than glass.
[0046] If elastically yielding plastic material is used for the
vessel walls and/or the cover, the sealing can be achieved by
resilient pressing of a sealing edge, as is indicated in FIG. 3.
The walls 4 of the vessel, made of glass or plastic for example,
extend parallel to one another and perpendicular to the base 8, for
example, cylindrically. The cover 2 is made of an elastic plastic.
Jutting upwards from its preferably flat cover base 7, there is a
collar 11 which, in the lower area 12, extends approximately
parallel to the walls 4 and, with respect to the latter, has no
clearance space or only a slight clearance space. The upper edge 13
of the collar 11 has a slightly greater diameter so that it is
elastically compressed upon insertion into the wall 4 and thus
bears tightly and with prestressing against the inner surface of
the wall 4 about its entire circumference. The position of the
cover in the vessel is secured by means of its friction on the
vessel walls.
[0047] In the illustrative embodiment according to FIG. 4, the
collar 11 of the cover likewise adjoins the walls 4 of the vessel
with only a slight gap width. At the free edge, the collar 11 and
the walls 4 are provided with a cooperating thread 25. A sealing
ring 14 is placed between the end faces of these parts. The ring 39
placed between the base plate 3 and the cover base 7 is discussed
in detail below.
[0048] Instead of the screw-on closure, it is possible to provide
any type of snap closure, or a clamp acting from outside on the
cover and holding it in its position. A design with a snap closure
is illustrated in FIG. 5. The edge 15 of the cover base 7 sits
sealingly on a shoulder 16 of the wall 4. It is held in this
position by virtue of the fact that the free edge 17 of the collar
11 of the cover 2 cooperates in a locking manner with a
circumferential nose 18 on the free edge of the vessel walls. The
shoulder 16 also has the advantage that it forms a limit stop,
which ensures the desired spacing between the cover base 7 and the
vessel base 3.
[0049] As is shown in FIG. 1, the cover base 7 can be shaped in
such a way that the opening 6 begins at an elevated part of the
base. This makes it easier to eliminate air bubbles without leaving
any behind. In general, however, this is not necessary. Remaining
air inclusions can be flushed out with an excess of the culture
medium or the reaction liquid.
[0050] In the examples according to FIGS. 1, 3, and 4, the opening
6 is connected to an ascending tube 24 which is at least as high as
the outflow section for removing air from the gap between the cover
collar 11 and the vessel walls 4. By means of the static pressure
in the ascending tube, it is possible, if so desired, to ensure
that air inclusions are driven out from the annular gap between the
circumferential surface of the cover and the internal surface 21 of
the vessel walls. This objective can also be achieved by giving the
cover opening such a high flow resistance with respect to liquid,
and by inserting the cover so quickly, that the overpressure
produced in the vessel guarantees the removal of any air from the
annular gap. FIG. 5 shows that the opening 6 narrows towards the
top to form a gap 23 of small width, which as a throttle has a
correspondingly high flow resistance. This gap can be designed in
such a way that, upon insertion of the cover into the vessel, it
opens in the manner of a nonreturn valve, with elastic deformation
of the material, to allow the escape of the air and excess liquid
and, thereafter, under the elastic restoring force of the material
and/or under the action of a clamp (not shown in the drawing), once
again closes in an airtight manner. By contrast, FIG. 4 shows a
stopper 19 for closing the opening 6. It is also possible, however,
to close the opening by means of a droplet of oil.
[0051] If there is a danger of cells being swept out with the
displaced liquid, the cover opening 6 is closed by a screen 22, as
is indicated diagrammatically in FIG. 3.
[0052] A plurality of cover openings 6 can be provided if the
intention is to use the device as a bioreactor through which a
reaction medium is to pass. In such a case, at least one opening is
connected to a liquid admission line and at least one other opening
is connected to a liquid discharge line.
[0053] In some cases, it is possible to dispense completely with an
airtight closure between the cover edge and the vessel walls,
namely, if an innocuous atmosphere is guaranteed inside an
apparatus receiving the device and if this atmosphere is protected
from the external atmosphere by means of an appropriate closure of
the apparatus, which in this case replaces the closure provided on
the device itself.
[0054] The illustrative embodiments according to FIGS. 6 and 8 are
based on the illustrative embodiment according to FIG. 1, although
they could readily start from any other embodiment of the invention
described above. They illustrate the formation of a narrower
chamber 30 inside a wider vessel 1. The walls 31 forming the
chamber 30 can involve a simple tube, for example, made of glass or
plastic, and in any case, sit with their lower ends on the top 8 of
the base plate 3 in order to separate the chamber 30 from its
surroundings. If the wall 31 is not designed in one piece with the
base plate 3, it can be attached thereto by means of a suitable
seal. The seal can be formed by an adhesive or, in accordance with
the example shown in FIG. 9, by an elastomer strip 32 placed firmly
and sealingly on the lower edge. In some cases it suffices if the
lower edge of the wall 31 is placed on the top 8 of the base 3
without an additional sealing means. In this case, and also when
using an elastomer seal which is not adhesively bonded, it is
expedient to promote the tight contact on the top 8 of the base by
means of the wall 31 being prestressed against the base 3. When it
is made in one piece with the cover 2, as in FIG. 7, this
prestressing can be produced by utilizing the elastic properties of
the vessel or cover, by means of the fact that the length of the
wall 31 has a certain over-dimension with respect to the base
spacing of the cover and the cover is placed firmly on the vessel
1, for example, with the aid of a screw-on, friction-type or snap
closure. However, such prestressing can also be produced if, as in
the illustrative embodiment according to FIG. 6, the wall 31 is
held releasably in a recess or holder 33 of the cover 2, with a
certain frictional force being exerted on the wall 31 by the holder
33. The holder 33 expediently encloses the wall 31 tightly. It is
formed, for example, by a bore in which the tubular wall 31 is
guided with a close fit. As FIG. 11 shows, this bore can be formed
by a cover opening 6.
[0055] If the cover 2 and the wall 31 are separate parts, they must
be positioned exactly in relation to one another in the vessel 1.
This is most easily done by first inserting the wall 31 into the
cover 2 and then inserting the latter, together with the wall, into
the vessel 1. Alternatively, the cover is first put into place and
the wall 31 is then inserted through the cover. If it is necessary
to install the chamber 30 inside the vessel 1 before the cover 2 is
put into place, it is best to use a positioning gauge for the wall
31, the dimensions of which positioning gauge in relation to the
vessel 1 are the same as those of the cover 2. This positioning
gauge can be removed before the cover 2 is inserted, for example,
after an adhesive connecting the wall 31 to the top 8 of the base
has hardened, or the positioning gauge remains in place underneath
the cover 2 when the latter is inserted. In this case, the
positioning gauge does not need to be a separate part; rather, it
can be structurally connected to the wall 31, for example, by means
of the latter having a series of arms protruding all around from
it, parallel to the base 3 of the vessel 1 or to the base 7 of the
cover, which arms terminate in positioning contact against the
internal surface 21 of the vessel walls 4.
[0056] In the same way as the vessel 1 is provided with a cover 2
equipped with an opening 6, the chamber 30 can be provided with a
closure member 34 which is suitable for limiting the height of the
substrate above the vessel base 8 and which contains a channel 35
which in turn can be closed by means of a stopper (not shown).
[0057] A further example of compartmentalization is illustrated in
FIG. 4. A sealing ring 39 of elastomer material, for example, a
commercially available O-ring, is placed between the base plate 3
and the cover base 7. The ring material is so thick that, in the
end position, it bears with elastic deformation tightly against the
base plate 3, and the cover base 7. In this way a chamber is
created within the sealing ring, which chamber is separated off
from the area located outside the sealing ring. It is accessible
via the outflow opening 6 in the cover, or via any other opening
created especially for this purpose, in order to permit a treatment
of its contents, which differs from the treatment in the
surrounding area. The ring 39 can be placed loosely in the vessel 1
before the cover 2 is put into place, in which case care must be
taken to ensure that the area enclosed by it lies at the point
where the opening 6 is expected. In order to ensure communication
with the opening 6 and to simplify the handling, the ring can also
be firmly connected to the cover 2 permanently or temporarily, for
example, by means of an adhesive or a form-fitting holder (not
shown in the drawing).
[0058] The compartmentalization according to the invention makes it
possible, in individual reaction steps or in a plurality of
reaction steps, to do something inside the chamber 30 that is
different from what is being done outside it, but with the thermal
conditions remaining the same. The cultures can also be the same if
the chamber 30 is inserted only after the culture has been placed
in the vessel 1. For example, in one stage of a multi-stage
procedure, twice as much enzymes can be introduced inside the
chamber 30 as is introduced outside the chamber. Thereafter, the
chamber wall 31 is removed, and all the cells are once again
treated identically. If a plurality of such insert chambers 30 are
used in a vessel, the possible variations multiply accordingly.
[0059] If the cells in the vessel are to be subjected to
electroporation or electrofusion, metal electrodes (not shown in
the drawing) are provided in the cover and base. As is shown in
FIG. 10, the cells are then placed on a porous base 36 or a
membrane of a cell culture insert 37 between base 3 and cover 2 and
are exposed to an electric field.
[0060] In the embodiments shown in FIGS. 12 to 14, a plurality of
vessels with base 3 and walls 4 are combined in one piece. It is
possible for this to include more than the two vessels shown. A
common cover 40 is provided for a plurality of these vessels and it
cooperates sealingly with the walls 4 of the vessels, in one of the
ways explained above. In the embodiments according to FIGS. 12 and
13, an outflow opening 6 in the cover 40 is provided for each
vessel.
[0061] In the embodiment according to FIG. 12, the cover 40 has, on
its upper side, an upwardly projecting edge 41 which delimits a
trough 42 into which the cover openings 6 open. On the one hand,
this trough collects the liquid which flows off when the cover is
inserted into the vessel. On the other hand, and this is the more
important point, the trough can be used for adding liquid which is
to be transferred into the treatment chambers 43 in order to modify
the properties of the liquid which is present there and in which
the cells are situated. It is possible to fill and empty the trough
using a suitable known pipetting robot.
[0062] The liquid present in the trough 42 can move through the
openings 6 and into the chambers 43 by diffusion. A common trough
42 for a plurality of openings 6 of different treatment chambers 43
is chosen if the liquid in the different treatment chambers 43 is
to be modified in the same way.
[0063] If the treatment liquid in adjoining treatment chambers 43
is to be modified in a different way, separate troughs are used.
For example, in the embodiment according to FIG. 12, a further wall
41 (not shown) could also be provided on the upper side of the
cover in the middle between the mouths of the opening 6. The
embodiment according to FIG. 13 achieves this aim in a slightly
different way. A depression 44 in the base of the trough 42 is
provided in the mouth area of each of the openings 6. Different
liquids can be introduced into the depressions 44 and then in each
case diffuse into the associated treatment chambers 43. In the
claims, the depressions 44 are also referred to as a trough.
[0064] The troughs make it possible to automate the process steps
for exchanging the treatment liquid. For this purpose, a suitable
machine (for example a known pipetting robot) is provided with
nozzles, indicated diagrammatically at 45, which are controlled by
a program in such a way that at a given time they introduce a
liquid of a desired type and amount into corresponding troughs.
[0065] If exchange of the treatment liquid by diffusion takes too
long, the treatment liquid can also be exchanged by forced flow.
Two illustrative embodiments of devices, which are suitable for
this, are shown in FIGS. 14 to 16.
[0066] In FIG. 14, it has been assumed (as in FIGS. 12 and 13) that
a plurality of vessels with treatment chambers 43 are combined in
one piece and are provided with a common cover 46. Each part of the
cover 46 allocated to a vessel has two openings 6a, 6b which open
into a trough 42 on the upper side of the cover. At least the
opening 6a is provided at the upper end with a depression 44. If so
desired, the cover can also be used for exchanging the treatment
liquid by diffusion, but it is mainly suited for forced exchange of
the treatment liquid. First, the new treatment liquid to be added
is introduced into the depression 44 of the opening 6a by means of
a nozzle 45 (which may belong to a pipette robot). The treatment
liquid present in the treatment chamber 43 is then suctioned off by
means of a pipetting robot or by means of a suction nozzle 48
lowered onto the other opening 6b, by which means the new liquid to
be added is at the same time drawn into the treatment chamber free
from bubbles. The amount suctioned off is precisely dosed to
guarantee exchange. The new liquid to be introduced can be present
in excess if the aim is to achieve as complete as possible a
replacement of the liquid previously present in the treatment
chamber 43, and if this liquid is to be flushed out by the
replacement liquid.
[0067] The illustrative embodiment according to FIGS. 15 and 16
permits flow exchange of the liquid without mechanical means. The
opening 6a, through which new liquid is to be delivered, is
connected at the top to a trough 47 of small cross section and
comparatively great height, while the opening 6b, through which the
liquid to be replaced is expelled, communicates with the trough 42
of large cross section and comparatively low height. If, as is
shown in FIG. 15, the high trough 47 is filled with the new liquid
to be added, which is shown by dots, the static pressure difference
in the troughs 47 and 42 results in the expulsion of the liquid
present in the treatment chamber 43, and symbolized by small
circles. According to FIG. 16, the exchange is completed when the
levels in troughs 42 and 47 are equal.
[0068] It will be appreciated that more than two openings can be
provided. For liquid exchange which is as complete as possible, it
may be expedient to provide the opening 6a for the new liquid to be
added in a central position and to distribute a greater number of
outlet openings uniformly near the circumference.
[0069] The exchange of liquid can also be carried out in the
reverse order by means of the fact that the new liquid to be
introduced is introduced at excess pressure into one of the
openings and the liquid to be replaced is thereby displaced from
the other opening. In this case, it may be expedient to design the
outlet openings, corresponding to the example in FIG. 5, so as to
open under excess pressure and to automatically reclose.
* * * * *