U.S. patent application number 17/230669 was filed with the patent office on 2022-03-10 for system for propagating cells.
The applicant listed for this patent is INSPHERO AG. Invention is credited to Jens Kelm, Jan Lichtenberg, Wolfgang Moritz, Bjoern Niggemann.
Application Number | 20220073851 17/230669 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073851 |
Kind Code |
A1 |
Niggemann; Bjoern ; et
al. |
March 10, 2022 |
System For Propagating Cells
Abstract
Provided is a system and a method for propagating cells, the
system comprising a multi well plate and a plate sealing means for
sealing at least one well of a multiwell plate, the plate sealing
means comprising at least one solid bulge or at least one bulge
comprising a solid base, said at least one bulge consisting of a
resilient elastomer to securely seal at least one well of the
multiwell plate. Also disclosed is the use of such a system for
propagating cells and the corresponding method.
Inventors: |
Niggemann; Bjoern;
(Schlieren, CH) ; Lichtenberg; Jan;
(Unterengstringen, CH) ; Moritz; Wolfgang;
(Bassersdorf, CH) ; Kelm; Jens; (Zurich,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSPHERO AG |
Schlieren |
|
CH |
|
|
Appl. No.: |
17/230669 |
Filed: |
April 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15566994 |
Oct 16, 2017 |
10982180 |
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PCT/EP2016/058390 |
Apr 15, 2016 |
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17230669 |
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International
Class: |
C12M 1/32 20060101
C12M001/32; C12M 1/00 20060101 C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 16, 2015 |
GB |
1506445.4 |
Claims
1.-20. (canceled)
21. A system for propagating cells, said system comprising a
multiwell plate and a plate sealing means for sealing a plurality
of wells of the multiwell plate, wherein the plate sealing means is
made of a resilient elastomer, wherein at least two neighbouring
wells of the multiwell plate are in fluid connection with one
another by means of at least one channel connecting said two
wells.
22. The system according to claim 21, wherein the plate sealing
means comprises at least two openings arranged at positions of said
at least two wells that are in fluid connection with one another,
thereby providing an inlet opening and an outlet opening present in
the multiwell plate system.
23. The system according to claim 22, wherein the inlet opening and
the outlet opening are configured as bores through the plate
sealing means.
24. The system according to claim 21, wherein the plate sealing
means comprises a resilient mat made of a gas-permeable, antistatic
silicone rubber.
25. The system according to claim 21, wherein the plate sealing
means is a planar mat comprised in a lid that further comprises a
plate and a circumferential brim.
26. The system according to claim 25, wherein said circumferential
brim has latches at opposite sides of the lid for engaging in
grooves of the multiwell plate, and wherein said plate has through
bores which align with the wells of the multiwell plate upon being
mounted thereto.
27. The system according to claim 26, wherein each of the wells of
the multiwell plate comprises a circumferential edge protruding
from the upper face of the multiwell plate.
28. The system according to claim 21 any one of the preceding
claims, wherein the wells of the multiwell plate comprise a central
deepening in the middle of the bottom of the well, defining a
microtissue culture compartment.
29. The system according to claim 21, wherein at least one of the
inlet opening and the outlet opening is provided with a funnel or
chimney.
30. The system according to claim 21, wherein at least a portion of
the wells of the multiwell plate is provided with an ultra-low
attachment surface.
31. The system according to claim 21, wherein the resilient
elastomer has an oxygen permeability of more than 1*109
cm3*cm/(s*cm2*cmHg).
32. The system according to claim 21, wherein the resilient
elastomer is selected from the group consisting of silicone rubber,
dimethylsilicone rubber vinyl methyl siloxane, phenyl vinyl methyl
siloxane, fluorosilicone rubber, nitrile rubber and natural
rubber.
33. The system according to claim 21, further comprising means for
controlling the temperature within the wells.
34. The system according to claim 21, further comprising a
temperature data logger as a portable measurement instrument.
35. A process for propagating cells, the method comprising the
steps of: providing a multiwell plate; introducing a suspension
comprising at least one cell into at least one well of the
multiwell plate; sealing at least the well of the multiwell plate
bearing the suspension containing at least one cell with a plate
sealing means defined in claim 21.
36. The process according to claim 35, wherein the plate sealing
means comprises at least two openings arranged at positions of said
at least two wells that are in fluid connection with one another,
thereby providing an inlet opening and an outlet opening present in
the multiwell plate system.
37. The process according to claim 35, further comprising
propagating the cells at a temperature of between 4.degree. C. and
37.degree. C.
38. The method of claim 37 or 38, further comprising continuously
or discontinuously replacing culture medium.
39. A process of propagating cells comprising providing and
propagating said cells in at least one well of a multiwell plate of
a system according to claim 21.
40. A pharmacological test assay comprising cultivating cells in a
system according to claim 1 and testing the toxicity of a drug or a
chemical compound on said cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/566,994, filed Oct. 16, 2017, which claims the priority
benefit of UK application 1506445.4, filed Apr. 16, 2015, each of
which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates in general to the field of cell
cultures. More particularly, the invention concerns a system for
propagating living cells, and methods for propagating living cells
using said system.
BACKGROUND
[0003] For analyzing biological mechanisms and for testing toxicity
of drugs or chemical compounds, the use of cells and microtissues
becomes increasingly admired. Usually, mammalian cells are
cultivated in form of a two dimensional monolayer of cells adhering
to a surface of or within the vessel used of cultivating said cell.
However, the natural in situ environment of a cell in a living
mammalian organism has a three-dimensional architecture. Thus,
cultivating mammalian cells in a three-dimensional architecture
leading to the formation of spheroids or microtissues provides test
systems which more closely resemble the morphological and
functional characteristics of a natural environment than a
monolayer of cells.
[0004] Generating spheroids or microtissues is cumbersome,
resource-demanding and fiddly. For this reason, spheroids and
microtissues are usually generated in facilities possessing the
know-how of propagating 3D cell cultures. For use, the spheroids or
microtissues are shipped from where they are generated to the site
of their use, for example, in drug screening assays. Typically,
cells in culture are cooled down to about 4.degree. C. or are even
frozen for being shipped, regardless of whether the cells are
present in a two-dimensional or in a three-dimensional
architecture. The cooling or freezing of the cells for and during
their shipment reduces their metabolism and increases their
survival rate. However, at their site of use, the cells have to be
thawed and/or warmed up to the temperature at which the subsequent
assay is performed. For example, mammalian cells being propagated
at 37.degree. C. will be frozen, shipped in liquid nitrogen or dry
ice, and warmed up to 37.degree. C. again in such a procedure.
[0005] Regardless of how gentle the cooling/freezing and
thawing/warming is performed, the viability of the cells within the
spheroid/microtissue is affected as the accuracy and/or reliability
of the subsequent assay is. Therefore, there is a need for
procedures and means which enable propagation and shipping of
cells, in particular in form of spheroids or microtissues, without
impairing the cell's viability.
[0006] Moreover, it is desired to keep the extent of manipulation
of spheroids and microtissues during propagation prior to an assay
to be performed as well as during the assay as small as possible.
Any transfer of spheroids and microtissues from one culture vessel
to another culture vessel might affect their integrity and/or
functionality. In addition, manipulating cell cultures always
renders the cell cultures prone to contamination with unicellular
microorganisms such as bacteria or yeasts when the lid of the
culture vessel is opened, for example for changing culture
media.
[0007] Therefore, there is a need for means for culturing cells, in
particular in form of microtissues or spheroids, which prevent
contamination to the utmost extent.
[0008] Mammalian cells may be cultivated in standard SBS/ANSI
format multiwell plates which are extensively used in molecular
biology laboratories. Multiwell plates are typically rectangular
flat plates comprising an array of wells. The precise dimensions
(length.times.width.times.height) of a multiwall plate of the ANSI
(American National Standard Institute) as recommended by the SBS
(Society for Biomolecular Screening) are 127.76 mm.times.85.48
mm.times.14.35 mm. Multiwell plates come in a variety of formats
within said base area but may have a different height. Typical
multiwell plates comprise [0009] 6 wells in a 2.times.3 array, each
of the wells including a volume of 2 ml to 5 ml; [0010] 12 wells in
a 3.times.4 array, each of the wells including a volume of 2 to 4
ml; [0011] 24 wells in a 4.times.6 array, each of the wells
including a volume of 0.5 ml to 3 ml; [0012] 48 wells in a
6.times.8 array, each of the wells including a volume of 0.5 to 1.5
ml; [0013] 96 wells in a 8.times.12 array, each of the wells
including a volume of 0.1 to 0.3 ml; or [0014] 384 wells in a
16.times.24 array, each of the wells including a volume of 0.03 to
0.1 ml.
[0015] The multiwell plates are available in different well formats
having either a flat bottom (F-bottom), a flat bottom with minimal
rounded edges to the wall (C-bottom), tapered walls (V-bottom) or a
bottom in U-shape (U-bottom). Multiwell plates are typically made
of polystyrene or polyvinyl chloride. For cell cultures, the
surfaces of these products are modified using an oxygen plasma
discharge to make their surfaces more hydrophilic so that it
becomes easier for adherent cells to grow on the surface which
would otherwise be strongly hydrophobic.
[0016] Multiwell plates may comprise wells having a circular cross
section or wells having a rectangular cross section, preferably a
square cross section.
[0017] Multiwell plates may be utilized to generate and propagate
spheroids or microtissues. For example, multicellular tumor
spheroids (MCTS) may be generated in that equal volumes of a
suspension of tumor cells is pipetted into agarose-coated wells of
a multiwell plate. At the lowest area of the agarose-coating of
each well only one spheroid forms, and all spheroids within the
multiwell plate will have approximately the same volume. In another
approach, microtissues or spheroids may be generated and propagated
in hanging drops, wherein specific multiwell plates are used.
[0018] During generating and/or propagating spheroids or
microtissues in a multiwell plate, for and during storage of
multiwell plates containing spheroids or microtissues, as well as
for and during shipping multiwell plates containing spheroids or
microtissues, the multiwell plates have to be covered. Said
covering prevents the cell culture in each well from contamination
and secures integrity of the cell culture. Conventional polystyrene
lids for multiwell plates are not suitable for shipping multiwell
plates containing spheroids or microtissues at ambient temperature,
because these conventional lids can not prevent spilling of the
well's content. For avoiding any spill, each of the wells of a
multiwell plate bearing cells has to be securely sealed.
[0019] For sealing multiwell plates that are used in molecular
biological methods such as--for example--Polymerase Chain Reaction
(PCR) experiments, a number of ways for sealing the plates are
known. For example, a foil or plastic film may be applied across
the entire upper surface of the plate. Heat sealable aluminum foils
provide an efficient gas and liquid tight seal, but they are
tiresome to apply and remove.
[0020] Certain adhesive plastic films made of polyethylene plates
may provide an alternative for sealing multiwell allowing exchange
of oxygen, carbon dioxide and water vapor. However, these films are
tiresome to apply and to remove. In addition, these films are
neither autoclavable nor reusable. Beyond that, the inventors found
that sealing multiwell plates bearing spheroids or microtissues
with an adhesive polymer film did not prevent substantial loss of
viability when the microtissue-bearing multiwell plates were kept
at a temperature of between 20.degree. C. and 37.degree. C.
compared to conventional lids for multiwell plates.
[0021] Surprisingly, the inventors found that loss of viability of
the cells of microtissues kept at a temperature of between
20.degree. C. and 37.degree. C. is significantly reduced, if the
multiwell plate containing the microtissues is to sealed with a
specifically configured plate sealing means made of a resilient
elastomer. Moreover, said plate sealing means improves handling of
microtissue cultures.
SUMMARY OF THE INVENTION
[0022] In a first aspect, the invention concerns a system for
propagating cells, wherein said system comprises a multiwell plate
and a corresponding plate sealing means for sealing at least one
well of said multiwell plate.
[0023] In a second aspect, the invention concerns a method for
propagating microtissues by using the system according to the first
aspect.
[0024] In a third aspect, the invention concerns the use of the
system according to the first aspect for propagating
microtissues.
[0025] In another aspect, the invention concerns the use of the
system in an assay.
[0026] In another aspect, the invention concerns a method for
investigating the effect of an analyte on living cells.
[0027] In a further aspect, the invention concerns the multiwell
plate of the system according to the first aspect and its use.
[0028] In yet another aspect, the invention concerns the plate
sealing means of the system according to the first aspect and its
use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows a plane view of an embodiment of the system for
propagating cells.
[0030] FIG. 2 shows a cross-sectional view along line C-C of the
embodiment shown in FIG. 1.
[0031] FIG. 3 shows an enlarged view of the encircled section
within the cross-sectional view of the embodiment shown in FIG.
2.
[0032] FIG. 4 shows a partial cross-sectional view of a plate
sealing means according to prior art and a well of a corresponding
multiwell plate.
[0033] FIG. 5 shows a partial cross-sectional view of an embodiment
of the plate sealing means of the invention and two adjacent wells
of a corresponding multiwell plate.
[0034] FIG. 6 shows a perspective view of an embodiment of a plate
sealing means of an embodiment of the system for propagating
cells.
[0035] FIG. 7 shows a cross-sectional view along line A-A of the
embodiment shown in FIG. 6.
[0036] FIG. 8 shows a cross-sectional view along line B-B of the
embodiment shown in FIG. 6.
[0037] FIG. 9 shows an enlarged view of the encircled section
within the cross-sectional view of the embodiment shown in FIG.
8.
[0038] FIG. 10 shows a cross section through a portion of an
embodiment of the system.
[0039] FIG. 11 shows a cross section through a portion of another
embodiment of the system.
[0040] FIG. 12 shows a cross section through a portion of a further
embodiment of the system.
[0041] FIG. 13 shows a cross section through a portion of yet
another embodiment of the system.
[0042] FIG. 14 is a cross sectional view of another embodiment of a
system for propagating cells.
[0043] FIG. 15A-15C show various embodiments of bulges of a plate
sealing means in cross sectional views.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0044] The present invention will be described with respect to
particular embodiments and with reference to certain drawings but
the invention is not limited thereto but only by the claims. The
drawings described are only schematic and are non-limiting. In the
drawings, the size of some of the elements may be exaggerated and
not drawn on scale for illustrative purposes. The dimensions and
the relative dimensions do not correspond to actual reductions to
practice of the invention.
[0045] Furthermore, the terms first, second and the like in the
description and in the claims, are used for distinguishing between
similar elements and not necessarily for describing a sequence,
either temporally, spatially, in ranking or in any other manner. It
is to be understood that the terms so used are interchangeable
under appropriate circumstances and that the embodiments of the
invention described herein are capable of operation in other
sequences than described or illustrated herein.
[0046] It is to be noticed that the term "comprising", used in the
claims, should not be interpreted as being restricted to the means
listed thereafter; it does not exclude other elements or steps. It
is thus to be interpreted as specifying the presence of the stated
features, integers, steps or components as referred to, but does
not preclude the presence or addition of one or more other
features, integers, steps or components, or groups thereof. Thus,
the scope of the expression "a device comprising means A and B"
should not be limited to devices consisting only of components A
and B. It means that with respect to the present invention, the
only relevant components of the device are A and B.
[0047] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, the particular features, structures or characteristics
may be combined in any suitable manner, as would be apparent to one
of ordinary skill in the art from this disclosure, in one or more
embodiments.
[0048] Similarly it should be appreciated that in the description
of exemplary embodiments of the invention, various features of the
invention are sometimes grouped together in a single embodiment,
figure, or description thereof for the purpose of streamlining the
disclosure and aiding in the understanding of one or more of the
various inventive aspects. This method of disclosure, however, is
not to be interpreted as reflecting an intention that the claimed
invention requires more features than are expressly recited in each
claim. Rather, as the following claims reflect, inventive aspects
lie in less than all features of a single foregoing disclosed
embodiment. Thus, the claims following the detailed description are
hereby expressly incorporated into this detailed description, with
each claim standing on its own as a separate embodiment of this
invention.
[0049] Furthermore, while some embodiments described herein include
some but not other features included in other embodiments,
combinations of features of different embodiments are meant to be
within the scope of the invention, and form different embodiments,
as would be understood by those in the art. For example, in the
following claims, any of the claimed embodiments can be used in any
combination.
[0050] Furthermore, some of the embodiments are described herein as
a method or combination of elements of a method that can be
implemented by a processor of a computer system or by other means
of carrying out the function. Thus, a processor with the necessary
instructions for carrying out such a method or element of a method
forms a means for carrying out the method or element of a method.
Furthermore, an element described herein of an apparatus embodiment
is an example of a means for carrying out the function performed by
the element for the purpose of carrying out the invention.
[0051] In the description provided herein, numerous specific
details are set forth. However, it is understood that embodiments
of the invention may be practiced without these specific details.
In other instances, well-known methods, structures and techniques
have not been shown in detail in order not to obscure an
understanding of this description.
[0052] The invention will now be described by a detailed
description of several embodiments of the invention. It is clear
that other embodiments of the invention can be configured according
to the knowledge of persons skilled in the art without departing
from the true spirit or technical teaching of the invention, the
invention being limited only by the terms of the appended
claims.
[0053] According to the first aspect, the invention provides a
system for propagating cells, wherein the system comprises a
multiwell plate and a plate sealing means for sealing at least one
well of the multiwell plate, such that cells within the at least
one well of the multiwell plate can be propagated therein when the
plate sealing means is applied to the multiwell plate.
[0054] The term "cells" as used herein refers to one or more living
cells, either prokaryotic cells or eukaryotic cells. The eukaryotic
cells may be of fungal, plant or animal origin. The term "cells"
includes human cells, i.e. cells of human origin. The term "cells"
comprises cells being present in form of individual cells, in form
of a monolayer of cells and also cells in form of a spheroid or
microtissue.
[0055] The term "microtissue" refers to an assembly of cells
attached to one another. Said assembly of cells may either be
present in form of a monolayer of cells or in form of a three
dimensional structure. Hence, the term "microtissue" as used herein
also comprises spheroids.
[0056] The term "propagating" with respect to cells and/or
microtissues refers to all aspects of cultivating cells. The term
"propagating" as used herein comprises the cultivation of cells
including their multiplication, development, proliferation and
maturation. Thus, "propagating" also comprises the formation of
microtissues. The term "propagating" as used herein also comprises
the maintenance, storage and/or shipping of cells and/or
microtissues in culture.
[0057] In an embodiment, the multiwell plate of the system is a
typical standard SBS/ANSI format multiwell plate, preferably a
multiwell plate as described herein above, more preferably a
multiwell plate comprising 96 wells in a 8.times.12 array or 384
wells in a 16.times.24 array.
[0058] In an additional and/or alternative embodiment, at least a
portion of at least one well of the multiwell plate, preferably at
least a portion of all wells of the multiwell plate, is/are
provided with an ultra-low attachment surface. Propagating cells in
a well which is provided with an ultra-low attachment surface
maintains the cells in a suspended, unattached state. Propagating a
spheroid or microtissue in a well that is provided with an
ultra-low attachment surface maintains integrity of the spheroid or
microtissue, and prevents the spheroid or microtissue from
undesired attachment-mediated maturation or differentiation,
including an undesired alteration in cell division properties.
[0059] Providing a well of a multiwell plate with an ultra-low
attachment surface can be done in various different ways. An
example of providing a well with an ultra-low attachment surface is
covalently binding a hydrogel layer to the substrate such as
polystyrene, said hydrogel being hydrophilic and neutrally charged.
Multiwell plates comprising such an ultra-low attachment surface
are commercially available from Corning Inc. Another example of an
ultra-low attachment surface is a surface made of a polymer
consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC). Such
surfaces are available from NOF America Corporation under the
tradename Lipidure.RTM..
[0060] Thus in an additional and/or alternative embodiment, at
least a portion of at least one well of the multiwell plate,
preferably at least a portion of all wells of the multiwell plate
comprises a coating layer consisting of a hydrogel such as agarose,
or a coating layer consisting of 2-methacryloyloxyethyl
phosphorylcholine (MPC).
[0061] In an additional and/or alternative embodiment, at least two
neighboring wells of the multiwell plate are in fluid
communication. In a preferred embodiment, the wells of a row of
wells within the multiwell plate are in fluid communication with
each other. For example, in a 96 well multiwell plate the 12 wells
of up to 8 rows of wells or 8 wells of up to 12 rows of wells can
be in fluid communication. In an alternative embodiment, all wells
of a multiwell plate are in fluid communication with each other.
Preferably, the wells of a row of wells or the wells of the
multiwell plate are in fluid communication with each other in that
each neighboring wells are in fluid communication.
[0062] In a preferred embodiment, the fluid communication between
at least two wells of the multiwell plate is established in that at
least one channel is provided between said at least two wells.
[0063] Preferably, the fluid communication between the two or more
wells of the multiwell plate is established by means of at least
one channel that is provided between two wells being in direct
fluid communication with each other. In alterative embodiments,
one, two, three, four or even more channels can be provided between
the two wells being in direct fluid communication with each other.
In embodiments comprising two or more channels between two wells
being in direct fluid communication with each other, said channels
run in parallel.
[0064] The diameter of the at least one channel and/or the number
of channels between two wells can be chosen to control the flow
rate of the fluid from one well to the other well. Generally, the
lower the number of channels and the smaller its/their diameter,
the smaller the flow rate of the medium that may migrate from one
well to the next well.
[0065] Providing a fluid communication between at least two wells
of the multiwell plate permits the system's use for assays wherein
the effect of a metabolite of product produced by the cells in one
well on the cells with the other well can be determined without any
need of opening the sealing of the wells and can also avoid the
need of aspirating culture medium from wells bearing cells.
[0066] The plate sealing means comprises at least one bulge
consisting of a resilient elastomer, wherein the bulge is
configured to securely fit into a well of the multiwell plate. The
term "resilient" means that the material is able to return to its
original shape after being pulled, stretched, pressed or bent
without permanent deformation or rupture. Thus, the bulge fits into
the opening of the well, and due to its resilience securely seals
the well. The term "securely sealing" means that the bulge does not
drop out of the well when the system is turned upside down, and
that no fluid inside the well leaks out of the sealed well when the
system is turned upside down.
[0067] In an additional and/or alternative embodiment, the at least
one bulge is a solid bulge. The at least one bulge may be
configured as a frustrum of a cone or as a frustrum of an inverse
cone. In an additional and/or alternative configuration, the at
least one bulge comprises a concave bottom for providing an
obstruse angle between the wall of the well and the bulge when the
bulge is appropriately inserted into said well. The term "bottom"
with respect to a bulge refers to the distal end of the bulge, i.e.
the end of the bulge being opposite of its base. In an additional
and/or alternative configuration, the at least one bulge comprises
a hollowed out bulge for improved flexibility and fit of said at
least one bulge into a corresponding well. The hollowing out of the
bulge provides a flexible edging of the bulge which improves fit of
the at least one bulge in the well and sealing of the well. In yet
an additional and/or alternative embodiment, the at least one bulge
comprises at least one flap. Preferably, the at least one bulge
comprises a single flap or a double flap. Said at least one flap is
circumferentially arranged at the outer edge of the distal portion
of the at least one bulge. The distal portion of the at least one
bulge is understood to be the portion opposite of the bulge's base.
Notwithstanding, the configurations comprising a concave bottom, a
hollow out and/or at least one flap comprise a base portion being
solid in its entire cross section.
[0068] In an additional and/or alternative embodiment, the
resilient elastomer is a silicone rubber.
[0069] A preferred silicone rubber has a hardness of Shore A in the
range of 55 to 65, measured according to DIN 53505.
[0070] A preferred silicone rubber has a specific gravity of
between about 1.13 and 1.17 g/cm3, measured according to DIN
53479.
[0071] A preferred silicone rubber has a tensile strength of about
7 MPa, measured according to DIN 53504 S2.
[0072] A preferred silicone rubber has an elongation of about 350%,
measured according to DIN 53504 S2.
[0073] In an additional and/or alternative embodiment, the
resilient elastomer is selected from the group consisting of
silicone rubber, preferably dimethylsilicone rubber vinyl methyl
siloxane, and phenyl vinyl methyl siloxane, fluorosilicone rubber,
and nitrile rubber and natural rubber.
[0074] In an additional and/or alternative embodiment, the
resilient elastomer is gas permeable. This is, that gas such
as--for example--air, oxygen and carbon dioxide--can permeate
through the silicone rubber. In an additional and/or alternative
embodiment, of the gas permeable resilient elastomer has an oxygen
permeability of more than 1*10.sup.9,
cm.sup.3*cm/(s*cm.sup.2*cmHg).
[0075] In an additional and/or alternative embodiment, the plate
sealing means is antistatic. This is, the silicone rubber material
of the plate sealing means reduces or even eliminates static
electricity.
[0076] In an additional and/or alternative embodiment, the plate
sealing means is autoclavable. That is, the plate sealing means can
be sterilized without being damaged or impaired in its
functionality as described herein by subjecting them to high
pressure saturated steam at 121.degree. C. for around 15 to 20
minutes.
[0077] In an additional and/or alternative embodiment, the plate
sealing means is reusable. This is, the plate sealing means can be
used multiple times for sealing a multiwell plate without being
damaged or impaired in its functionality.
[0078] In an additional and/or alternative embodiment, the plate
sealing means comprises at least one bulge comprising a resealable
septum. This is, the plate sealing means may be punctured by means
of a 18 gauge needle and reseals when the 18 gauge needle is pulled
out of the plate sealing means.
[0079] The properties such as gas-permeability is not significantly
altered upon puncturing.
[0080] In an additional and/or alternative embodiment, the plate
sealing means is non-adhesive. This is, the plate sealing means
does not have a tack.
[0081] In an additional and/or alternative embodiment, the at least
one bulge of the plate sealing means comprises a rim at its base,
i.e. where the bulge protrudes from the second face. The rim of the
at least one bulge is circumferential. The rim of the at least one
bulge is configured not to fit into a well of a corresponding
multiwell plate, but to sit on top of the well next to the well's
aperture.
[0082] The rim prevents the second face of the plate sealing means
from getting into contact with the upper face of the multiwell
plate. This configuration permits easier removal of the plate
sealing means from the multiwell plate compared to an embodiment
without such rims.
[0083] The at least one bulge may have a circular cross section, a
rectangular cross section of a square cross section. The sides of
the bulge are configured to narrow the cross section from the base
of the bulge towards the tip of the bulge. In an embodiment, the
opposite sides of a bulge include an angle of 30.degree. which
corresponds to an angle of 15.degree. between the longitudinal axis
of the bulge and the flange.
[0084] An angle of approximately 30.degree. between the sides of a
bulge permits easy insertion and removal of the bulge into/from a
well of a corresponding multiwell plate, but secures tight and
snugly fitting of the know within the well.
[0085] In an additional and/or alternative embodiment, the plate
sealing means is present in form of a resilient mat made of a
gas-permeable, antistatic silicone rubber. The plate sealing means
of this embodiment comprises a first face and a second face. The
first face constitutes the upper face of the plate sealing means
when the plate sealing means is in use, i.e. applied to a multiwell
plate. Thus, the first face of the plate sealing means faces away
from the multiwell plate when the plate sealing means is applied to
a multiwell plate. The second face of the plate sealing means is at
the opposite side of the first face of the plate sealing means and
constitutes the lower face, i.e. the face of the plate sealing
means facing the multiwell plate when the plate sealing means is in
use, i.e. applied to a multiwell plate.
[0086] In an embodiment, the first face has a flat or even surface
substantially free from dimples. In an additional and/or
alternative embodiment, the first face has labelling means for
better identifying those areas of the first face being aligned with
the wells of a multiwell plate when the plate sealing means is
applied to a multiwell plate. The labelling means may be an
embossed, imprinted or engraved structure. The labelling means may
comprise letters, numbers or combinations of letters and numbers
unique for each well, rims or walls indicating the position of each
well, wherein the labelling means are arranged on the first face of
the plate sealing means at positions corresponding to the wells of
the multiwell plate, i.e. opposite of bulges protruding from the
second face of the plate sealing means.
[0087] The plate sealing means in form of the resilient mat
comprises at least one bulge protruding from the second face of the
plate sealing means. Preferably the plate sealing means comprises
an array of bulges protruding from the second face of the plate
sealing means, wherein the array of bulges matches the array of
wells of a corresponding multiwell plate, i.e. of a multiwell plate
said plate sealing means is configured to seal. The at least one
bulge, preferably each bulge of the array of bulges, is/are sized
and shaped to sit firmly in a well of the corresponding multiwell
plate.
[0088] The at least one bulge or the bulges or the array of bulges
are solid elements of the plate sealing means protruding from the
second face of the plate sealing means or elements comprising a
solid base portion. That is, the bulges are solid or comprise a
solid base, and they consist of the silicone rubber, the plate
sealing means are made of such that the at least one bulge or the
bulges are integral elements of the plate sealing means such that
the thickness of the rubber material is increased in the area of
the bulges compared to the area between two bulges. The bulges are
not configured as dimples which are typically generated in that the
rubber film is deformed such that the thickness of the rubber
material in the area of the dimple is essentially the same as
outside of the dimple.
[0089] The bulges provide a secure sealing of the wells of a
corresponding multiwell plate when the plate sealing means is
applied to the multiwell plate, thus preventing any liquids from
escaping from or entering the well that is sealed. It is believed
that the tight sealing of the wells is based on the fact that the
bulge is solid or comprises a solid base rather than being a
dimple. The restoring force of the resilient bulge when inserted
into the well of the multiwell plate appears to be higher than the
restoring force of a dimple having the same shape and size.
Therefore, it is believed that the better sealing of a bulge
compared to a dimple accounts for the improvement in cell
viability.
[0090] Additional configurations of the bulge further improve their
efficacy in sealing a well and thereby securing propagation of
cells within the well. Such additional configurations are--for
example--a concave bottom of the bilge, a hollow out of the bulge
and/or at least one circumferential flap at the distal portion of
the bulge.
[0091] The plate sealing means comprising solid bulges or bulges
comprising a solid base is made of a resilient elastomer. Thus, a
flexible mat is provided that can be easily applied to a multiwell
plate, easily removed from the multiwell plate and easily
re-applied to the same or another multiwell plate, and
simultaneously provide a tight sealing of the wells of a multiwell
plate.
[0092] In an embodiment, the plate sealing means covers the entire
surface of the multiwell plate. In an alternative embodiment, the
plate sealing means does not cover the entire surface of the
multiwell plate, but only a portion of the surface.
[0093] The plate sealing means comprises a resilient silicone mat
which covers essentially the entire upper surface of a standard
96-well plate, and comprises 96 bulges such that every well of a
standard 96 well plate can be sealed using this embodiment of the
plate sealing means.
[0094] In an embodiment, the plate sealing means comprises a
circumferential brim. The brim extends perpendicular to the plane
of the mat and in the direction of the bulges. Thus when mounted to
a multiwell plate the brim of the plate sealing means encompasses
the sides of the multiwell plate. Thus the embodiment of the plate
sealing means has the form a conventional polystyrene lid for
multiwell plates, but is resilient.
[0095] The bulges preferably have a circular cross section for
securely fitting into wells having a circular cross section too. It
is understood that the bulges of the plate sealing means may be
configured as described herein before with respect to the
bulges.
[0096] The term "multiwell plate" as used herein refers to
rectangular flat plates comprising an array of wells, preferably
having dimensions as recommended by the Society for Biomolecular
Screening as set forth herein above.
[0097] The term "corresponding multiwell plate" as used herein
refers to a multiwell plate which has the same number of wells as
the plate sealing means has bulge, wherein the wells of the
multiwall plate are present in the same array as the bulges of the
plate sealing means are, and/or wherein the wells have the same
cross section as the bulges of the plate sealing means have, and
where the diameter of the well is identical to the diameter of the
bulge at about half of its height. The term "corresponding
multiwell plate" shall indicate that the plate sealing means is a
plate sealing means configured for sealing the wells of a specific
multiwell plate.
[0098] The term "cultivating" as used herein comprises maintaining
or propagating cells in vitro, wherein the term "maintaining"
comprises storage of cells at lower temperatures, for example at
about 4.degree. C., or even frozen, for example at about
-20.degree. C., about -80.degree. C. or even at about -180.degree.
C. The term "cultivating" comprises proliferation of cells in vitro
as well as the maintenance of cells in vitro, i.e. under conditions
where the cells multiply, multiply at a reduced speed, or do not
multiply.
[0099] The term "shipping" as used herein refers to freight
transport, i.e. the physical process of transporting commodities
and merchandise goods and cargo including living organs, tissues,
microtissues, spheroids and cells. The term "shipping" comprises
transport by sea, by land or air.
[0100] In an embodiment the plate sealing means is made of a
resilient elastomer, the resilient elastomer is preferably selected
from the group consisting of silicone rubber, preferably
dimethylsilicone rubber vinyl methyl siloxane, and phenyl vinyl
methyl siloxane, fluorosilicone rubber, and nitrile rubber and
natural rubber.
[0101] In an embodiment, the resilient elastomer of the plate
sealing means is gas permeable. This is, that gas such as--for
example--air, oxygen and carbon dioxide--can permeate through the
elastomer. Preferably, the resilient elastomer has an oxygen
permeability of more than 1*10.sup.9, cm.sup.3*cm/(s*cm.sup.2*cm
Hg).
[0102] A preferred silicone rubber has a hardness of Shore A in the
range of 55 to 65, measured according to DIN 53505.
[0103] A preferred silicone rubber has a specific gravity of
between about 1.13 and 1.17 g/cm3, measured according to DIN
53479.
[0104] A preferred silicone rubber has a tensile strength of about
7 MPa, measured according to DIN 53504 S2.
[0105] A preferred silicone rubber has an elongation of about 350%,
measured according to DIN 53504 S2.
[0106] The plate sealing means is antistatic. This is, the silicone
rubber material of the plate sealing means reduces or even
eliminates static electricity.
[0107] The plate sealing means is autoclavable. That is, the plate
sealing means can be sterilized without being damaged or impaired
in its functionality as described herein by subjecting them to high
pressure saturated steam at 121.degree. C. for around 15 to 20
minutes.
[0108] The plate sealing means is reusable. This is, the plate
sealing means can be used multiple times for sealing a multiwell
plate without being damaged or impaired in its functionality.
[0109] In an additional embodiment, the plate sealing means
comprises at least one bulge comprising a resealable septum. This
is, the plate sealing means may be punctured by means of a 18 gauge
needle and reseals when the 18 gauge needle is pulled out of the
plate sealing means. The properties such as gas-permeability is not
significantly altered upon puncturing.
[0110] The plate sealing means is non-adhesive. This is, the plate
sealing means does not have a tack.
[0111] In an additional embodiment, the at least one bulge of the
plate sealing means comprises a rim at its base, i.e. where the
bulge protrudes from the second face. The rim of the at least one
bulge is circumferential. The rim of the at least one bulge is
configured not to fit into a well of a corresponding multiwell
plate, but to sit on top of the well next to the well's
aperture.
[0112] The rim prevents the second face of the plate sealing means
from getting into contact with the upper face of the multiwell
plate. This configuration permits easier removal of the plate
sealing means from the multiwell plate compared to an embodiment
without such rims.
[0113] The at least one bulge may have a circular cross section, a
rectangular cross section of a square cross section. The sides of
the bulge are configured to narrow the cross section from the base
of the bulge towards the tip of the bulge. In an embodiment, the
opposite sides of a bulge include an angle of 30.degree. which
corresponds to an angle of 15.degree. between the longitudinal axis
of the bulge and the flange.
[0114] An angle of approximately 30.degree. between the sides of a
bulge permits easy insertion and removal of the bulge into/from a
well of a corresponding multiwell plate, but secures tight and
snugly fitting of the know within the well.
[0115] In an embodiment, the plate sealing means comprises a
circumferential brim. The brim extends perpendicular to the plane
of the mat and in the direction of the bulges. Thus when mounted to
a multiwell plate the brim of the plate sealing means encompasses
the sides of the multiwell plate. Thus the embodiment of the plate
sealing means has the form a conventional polystyrene lid for
multiwell plates, but is resilient.
[0116] The plate sealing means may additionally comprise posts or
pins fitting respectively into holes other than the wells within a
multiwell plate. The posts are shaped such that they fit into the
holes and contact the walls of the holes. The posts/holes are
further means preventing the plate sealing means from moving
laterally with respect to the multiwell plate, and prevent
misalignment of the plate sealing means, in particular when the
plate sealing means is only partially removed from the multiwell
plate, for instance when access to individual wells of the
multiwell plate is required.
[0117] In an embodiment, the plate sealing means comprise at least
one inlet opening and at least one outlet opening. In a preferred
embodiment, the plate sealing means comprises an inlet opening and
an outlet opening for each row of wells within the multiwell plate.
Preferably, each of the inlet openings of the plate sealing means
corresponds to the first well of the row of wells, whereas the
outlet well corresponds to the last well of the row of wells. Said
at least one inlet opening and said at least one outlet opening are
holes or through bores in the plate sealing means such that the
corresponding well of the multiwell plate is not sealed by the
plate sealing means when the plate sealing means is mounted to the
multiwell plate.
[0118] The inlet opening permits adding or supplementing culture
medium to the at least one well. The outlet opening permits removal
or aspiration of culture medium from the at least one well. In
combination with the embodiment of the multiwell plate comprising
at least two wells being in fluid connection, mounting of the plate
sealing means comprising at least one inlet opening and one outlet
opening permits supplementing all wells with culture medium that
are in direct or indirect fluid connection with inlet well.
[0119] In a further embodiment, the at least one inlet opening
and/or the at least one outlet opening of the plate sealing means
are configured as funnel or chimney. "Funnel" refers to a structure
having a diameter narrowing towards the point of connection to the
plane of the silicone mat, at least along of at least a portion of
its longitudinal direction. "Chimney" refers to a pipe or tube like
structure having a constant diameter along its longitudinal
direction.
[0120] In another embodiment, the plate sealing means is configured
in in form of a substantially planar resilient mat without bulges.
In this embodiment, the plate sealing means comprises a rigid
frame, preferably made of hard plastics such as polystyrene, and is
configured to be used as a lid for a corresponding multiwell plate.
Said rigid frame comprises a plate comprising bores, wherein said
bores align to the wells of a corresponding multiwell plate. Said
plate comprises an outer face and an inner face. The outer face
faces away from the multiwell plate, whereas the inner face faces
towards the multiwell plate upon mounting the plate sealing means
to the multiwell plate. The substantially planar resilient mat is
provided on the inner face of the plate.
[0121] In an additional and/or alternative embodiment, the system
comprises a quick lock system for securing the rigid frame to the
multiwell plate. In an embodiment, the quick lock system comprises
at least two locking latches at opposite ends of the frame. Said
latches engage with grooves or slots or trenches at the
corresponding positions at the outer circumference of the
corresponding multiwell plate. By forcing the frame onto the
multiwell plate, the latches engage and thereby not only secures
the frame onto the multiwell plate, but simultaneously sealing the
wells of the multiwell plate.
[0122] In a preferred embodiment of the multiwell plate for being
sealed by the plate sealing means of the latter embodiment, each
well comprises a circumferential edge at its outlet opening. Said
circumferential edge protruding from the upper face of the
multiwell plate in perpendicular direction thereto. Said
circumferential rim is pressed into the planar resilient mat when
the plate sealing means is mounted to the multiwell plate, thereby
providing the secure sealing if the well.
[0123] The substantially planar resilient mat is made of a
resilient elastomer as described herein before with respect to the
bulges.
[0124] Referring to FIGS. 1 to 3 an embodiment of the system (1)
for propagating microtissues is displayed. FIG. 1 shows a
perspective view on top of the system (1). The embodiment comprises
a 96-well multiwell plate (10) comprises a plurality of wells (11)
being arranged in a 12.times.8 array. The multiwell plate (10) is
covered with an embodiment of a plate sealing means (20, not shown
in FIG. 1).
[0125] The embodiment of the multiwell plate (10) comprises three
positioning means (12, 13, 14) in form of apertures. The three
positioning means (12, 13, 14) are arranged such that a lid or a
plate sealing means (20) comprising corresponding protuberances
(22, 23, 24) which are configured for engaging said positioning
means (12, 13, 14) can be applied to the multiwell plate (10) in
one specific orientation only.
[0126] The plate sealing means (20) comprises a plurality of bulges
(21) protruding from one face of the plate sealing means. The
plurality on bulges (21) are present in the same array as the wells
of the corresponding multiwell plate. In the embodiment shown in
FIGS. 1 to 3, the plurality of bulges (21) are arranged in a
12.times.8 array.
[0127] The presence of the positioning means (12, 13, 14) prevents
a plate sealing means (20) comprising a symmetrical array of bulges
(21) to the applied to the multiwell plate (10) in an inadvertent
orientation. Thereby avoiding contamination.
[0128] In addition, the embodiment of the plate sealing means (20)
shown in FIG. 2 comprises a circumferential brim (25) at the outer
border of the plate sealing means (20). The brim (25) extends the
sides of the plate sealing means (20) over the upper surface of the
multiwell plate (10) when the plate sealing means (20) is
appropriately mounted to the multiwell plate (10).
[0129] FIG. 3 illustrates how a bulge (21) of the plate sealing
means (20) snugly fits into the corresponding well (11) of a
corresponding multiwell plate (10) providing a tight seal of the
well (11) due to its size and resilience. The bulge (21) is
configured to leave a volume for cells, culture medium and/or air
in the well (11).
[0130] FIG. 4 shows a cross-sectional view of a part of a
commercially available plate sealing means (30). The view represent
a single well of the multiwell plate and the corresponding section
of a commercially available plate sealing means (30). The
commercially available plate sealing means (30) consists of a film
that has been subjected to an embossing process such that dimples
(31) are generated. Said dimples (31) are configured to fit into a
well (11) of a corresponding multiwell plate (10). The thickness of
the film in the areas of the dimples is the same as in the areas
next to the dimples.
[0131] Referring to FIG. 5 showing a cross-sectional view of a part
of an embodiment of a system according to the invention. The part
represents two neighboring wells (11) of a multiwell plate (10),
and the corresponding section of a corresponding plate sealing
means (20). The figure illustrates that the plate sealing means (20
comprises bulges (21) which are solid elements of the plate sealing
means (20) were the material of the plate sealing means (20) is
much thicker than in the region between the bulges (21).
[0132] The bulges (21) of the embodiment of the plate sealing means
(20) as shown in FIG. 5 comprise a rim (26) at the base of the
bulges (21), i.e. where the bulges (21) protrude from the plane of
the plate sealing means (20). Upon sealing of a well (11) of a
corresponding multiwell plate (10), the bulges (21) fit snugly into
the open end of the well (11) such that the rim (26) resides on the
upper face (16) of the multiwell plate (10). The rim (26) of each
bulge (21) is a circumferential element. The presence of said rims
(26) at each bulge (21) renders removal of the plate sealing means
(20) from the corresponding multiwell plate (10) after the plate
sealing means (20) being mounted to the multiwell plate (10) much
easier, thereby preventing undesired shaking which might affect
formation and/or integrity of a microtissue within a well of the
multiwell plate (10) and/or might lead to undesired spillover of
culture medium from one well to the other.
[0133] Referring to FIGS. 6 to 9 an embodiment of the plate sealing
means (20) is displayed. FIG. 6 shows a perspective view on top of
the second face (27) of the plate sealing means (20). The
embodiment of the plate sealing means (20) as shown comprises an
array of solid bulges (21) protruding from the second face (27).
The plurality of solid bulges (21) are arranged in an 8.times.12
array corresponding to the array of wells of a typical 96-well
plate.
[0134] FIG. 7 illustrates a longitudinal section along line A-A of
the plate sealing means (20) of FIG. 6, whereas FIG. 8 illustrates
a longitudinal section along line B-B of the plate sealing means
(20) of FIG. 6.
[0135] FIG. 9 is an enlarged view of portion II encircled in FIG.
8. FIG. 9 emphasis a solid bulge (21) of the plate sealing means
(20) which protrudes from the second face (27) of the plate sealing
means.
[0136] The solid bulges (21) protruding from the second face (27)
of the plate sealing means (20) preferably have a height of about
1.5 times the thickness of the mat, i.e. the distance measured from
the first face (28) to the second face (27) of the mat. The bulges
(21) comprise a base where they merge with the second face (27) of
the plate sealing means (20), and a tip. The sides (29) of each
bulge (21) are tapered such that the diameter of the bulge (21) at
its tip is smaller than the diameter at the base of the bulge (21).
The flanges (29) on opposite sides of the solid bulge (21) include
an angle (a) of about 30.degree. such that the bulge can be easily
inserted into a well of a corresponding multiwell plate, fits
snuggly into the well, and provides a tight seal of the well. The
sides are preferably tapered in that they include an angle (7) of
about 30.degree..
[0137] FIG. 10 illustrates a portion of a further embodiment of a
system, wherein the plate sealing means (20) comprises at least one
solid bulge (21) for sealing the corresponding well (11) of a
corresponding multiwell plate (10). The bulge (21) comprises a
self-sealing septum such that, for example, an injection needle
(40) can be poked through the self-sealing septum of the bulge
(21). Using a syringe or other suitable means, culture medium can
be aspirated trough the injection needle (40) from the well (11)
and/or added thereto without the need of removing the entire plate
sealing means (20) from the multiwell plate (10). Upon removal of
the injection needle (40), the septum seals itself such that the
plate sealing means (20) still provides a tight seal of the well
(11).
[0138] Also shown in FIG. 10 is an embodiment comprising a
specifically designed well (11) for propagating microtissues (2).
Said well (11) comprises a microtissue culture compartment (15)
which is present at the bottom of the well (11). Said microtissue
culture compartment (15) is a small partition in the middle of the
bottom of the well which is in fluid connection therewith and thus
constitutes an integral part of the well.
[0139] FIG. 11 illustrates a portion of another embodiment, wherein
the plate sealing means (20) comprising a bulge (21) with two
self-sealing septa. This embodiment permits inserting an inlet
conduit (41) and an outlet conduit (42) through the plate sealing
means (20) into the well (11). This configuration permits providing
a constant flow of culture medium through the well (11).
[0140] FIG. 12 is a schematic representation of another embodiment
of the system (1) for propagating microtissues. The multiwell plate
(10) is configured such that the neighboring wells (11, 11', 11'')
of one row of wells of the multiwell plate (10) are in fluid
connection with one another by means of at least one channel (17)
connecting the wells.
[0141] In addition, the embodiment comprises a plate sealing means
(20), comprising an inlet opening (210) and an outlet opening
(220). The inlet opening (210) is placed instead of the first bulge
of a row of bulges, whereas the outlet opening (220) is placed
instead of the last bulge of the row of bulges.
[0142] The inlet opening (210) and/or the outlet opening (220) may
be configured as through holes in the silicone mat. In an
alternative embodiment the inlet opening (210) and/or the outlet
opening (220) may be configured as a funnel or chimney which
comprises a wall (211, 221) extending substantially upright from
the first face (28) of the plate sealing means (20) when mounted to
the multiwell plate (10).
[0143] The open end of the inlet opening (210) and the open end of
the outlet opening may be sealed by means of a plug (43, 45). The
plug may be made of a plastic material such as a silicone rubber.
In an embodiment, the plug (43) may comprise a microporous section
(44). Alternatively, the plug (45) may consist of a microporous
material. Sealing the inlet opening and/or the outlet opening of
the plate sealing means with a plug consisting of or to comprising
a microporous material improves the exchange of gaseous fluids in
the system (1).
[0144] FIG. 13 shows a cross section through a portion of an
embodiment of the system comprising a multiwell plate (10) and a
plate sealing means (20). The multiwell plate comprises a plurality
of wells being in fluid communication with one another. Neighboring
wells (11) are put in fluid communication by means of one or more
channels (17) extending from one well (11) to the next well in a
row of wells.
[0145] In a preferred embodiment, the one or more channels
connecting neighboring wells is/are not disposed at the lower end
of the well, but at a distance in height from the bottom of the
wells.
[0146] The embodiments shown in FIGS. 12 and 13 allow to supply
culture medium through the inlet opening of the plate sealing means
mounted to the corresponding multiwell plate without the need of
removing the plate sealing means (20) from the multiwell plate
(10). The culture medium supplied to the first well, i.e. the well
underneath the inlet opening (210) will flow through the channels
connecting the wells with one another such that all wells being in
fluid communication with each other are supplied with culture
medium.
[0147] The embodiment further provides the opportunity of providing
a constant flow of culture medium through the wells being in fluid
communication if a constant supply of culture medium through the
inlet opening (210) and a constant removal of culture medium
through the outlet opening (220) is provided. The constant flow of
culture medium may be obtained by utilizing hydrostatic forces in
that the level of culture medium in the funnel of the inlet opening
is kept higher than the level of culture medium at the outlet
opening. The height difference thus provides a hydrostatic force
which forces the culture medium flowing through the channels from
one well to the next well towards the last well which is the well
underneath the outlet opening. The velocity of medium flow through
from one well to the next well can also be determined by the
diameter and/or number of channels connecting said wells.
[0148] Referring to FIG. 14, another embodiment of the system is
shown. The system (1) of this embodiment comprises a multiwell
plate (310) and a plate sealing means (320). The multiwell plate
(310) comprises wells (311, 311') including microtissue
compartments (315, 315'). The wells (311, 311') are in fluid
communication due to the channel (317) which is provided between
said wells (311, 311'). In addition, each of the wells (311, 311')
comprises a circumferential edge (325, 325') protruding from the
upper face of the multiwell plate (310). The multiwell plate
further comprises grooves (312, 312').
[0149] The embodiment further comprises a plate sealing means (320)
which is configured as a lid comprising a plate (326) and a
circumferential brim (322). Said brim (322) comprises latches (323,
323') at opposite sides of the lid for engaging in the grooves
(312, 312') upon correct mounting of the plate sealing means (320)
to the multiwell plate (310). The plate sealing means (320) further
comprises a planar mat (321) made of a resilient elastomer. Said
planar mat (321) may be attached to the plate (326). The plate
further comprises through bores (324, 324') which align with the
wells (315, 315') of the multiwell plate upon being mounted
thereto.
[0150] In an embodiment of the first aspect, the system further
comprises means for controlling the temperature within the wells of
the multiwell plate.
[0151] The means for controlling the temperature are configured to
control the temperature such that the temperature within the wells
of the multiwell plate is a temperature of between 25.degree. C.
and 37.degree. C.
[0152] In an alternative and/or additional embodiment, the means
for controlling the temperature within the wells of the multiwell
plate are configured to control the temperature during shipping of
the multiwell plate being sealed with a plate sealing means
according to the first aspect.
[0153] In another and/or alternative embodiment of the first
aspect, the system further comprises a temperature data logger. The
temperature data logger, also called temperature monitor, is a
portable measurement instrument that is capable of autonomously
recording the temperature over a defined period of time. The
digital data being recorded can be retrieved, viewed and evaluated.
The temperature data logger is used to monitor the temperature of
the cells being propagated in that the ambient temperature of the
cell culture is measured. Using the temperature data logger is
advantageous when the cells or microtissues are shipped. The
temperature data logger is included in the container bearing the
cell culture to be shipped within at least one sealed multiwell
plate. The temperature data logger is preferably placed in close
proximity to the at least one multiwell plate including the cells
or microtissues. The temperature data logger monitors the
temperature and alterations in temperature within the container and
thus the temperature said cells or microtissues are exposed to.
This temperature monitoring permits use of only those cells or
microtissues for any assay that were not exposed to undesired
temperatures or inadequate temperatures.
[0154] Referring to FIG. 15 a to c various embodiments of bulges
are shown schematically in cross-sectional views. FIG. 15 a shows
embodiments (I to IV) configures as a frustrum of a cone, wherein
the bulges comprise a concave bottom (101, 102, 103, 104).
Embodiments III and IV are configured as inverse frustrum of a
cone. FIG. 15 b shows embodiments (V to VIII) wherein the bulges
comprise a hollow out (111, 112, 113, 114). The bulges comprise a
solid portion at their base (121, 122, 123, 124) and a
circumferential rim (131, 132, 133, 134) at their distal portion.
FIG. 15 c shows embodiments of bulges comprising a single flap
(embodiments IX and X) of a double flap (embodiment XI). The single
flaps (141, 142) and the double flap consisting of two flaps (143
and 144) are configured at the outer wall of the bulge where they
extend outwards and circumferentially from the circumferential rim
of the bulge. In the embodiments shown, the flaps (141, 142 and
144) are arranged at the distal end of the bulge. The bulges as
shown comprise a solid base, a hollow out and a circumferential rim
as exemplified with respect to embodiment IX, wherein the solid
base (151) extends perpendicular from the second face (27) of the
plate sealing means (20). The bulge comprises a hollow out (161)
providing a circumferential rim (171) which further extends from
the plate sealing means.
[0155] Upon inserting a bulge comprising a concave bottom, a hollow
out and/or at least one rim into a corresponding well, the distal
portion of said bulge can better snuggle to the inner lining of the
wall of the well and thereby provide an even better sealing of the
well than a solid bulge.
[0156] It is understood that the dimensions of a bulge may vary
upon the dimension of the well to be sealed, and that bulges of
different heights may be provided.
[0157] According to the second aspect, the invention provides a
method of propagating cells, wherein cells are provided and
propagated in at least one well of a multiwell plate according to
the system, said at least one well of the multiwell plate being
sealed with a plate sealing means according of the system.
[0158] The method comprises the steps of: [0159] providing a
multiwell plate as described herein before; [0160] introducing a
suspension comprising at least one cell into at least one well of
the multiwell plate; [0161] sealing at least the well of the
multiwell plate bearing the suspension containing at least one cell
with a plate sealing means as described herein before.
[0162] In an embodiment of the method, said cells are eukaryotic
cells. In a further and/or additional embodiment, the cells are
mammalian cells. In yet a further and/or alternative embodiment,
the cells are human cells.
[0163] In an embodiment of the method, the cells are propagated,
propagating the cells at a temperature of between 4.degree. C. and
37.degree. C., preferably at a temperature of between 18.degree. C.
and 37.degree. C., more preferably at a temperature of between
25.degree. C. and 37.degree. C., and most preferably at a
temperature of between 29.degree. C. and 37.degree. C. for a period
of time. Alternatively at a temperature of between 18.degree. C.
and 27.degree. C., preferably at a temperature of between
21.degree. C. and 25.degree. C.
[0164] The temperature for cultivating and/or shipping cells is
preferably maintained constant by utilizing suitable means of
controlling the temperature.
[0165] In an embodiment of the method, the propagation comprises
providing an exchange of culture medium. Said exchange of culture
medium may be performed continuously or discontinuously.
[0166] In an embodiment, wherein the culture medium is
discontinuously exchanged in individual wells of the system, an
injection needle is poked through the plate sealing means,
preferably through a self-sealing septum of the bulge sealing said
well, and inserted into the well. If already present, the culture
medium in the well is aspirated through the needle, and fresh
culture medium is supplied to the well through the needle.
[0167] In an embodiment wherein the culture medium is continuously
exchanged in individual wells of the system, an inlet conduit and
an outlet conduit are inserted through the plate sealing means,
preferably through one or more self-sealing septa of the bulge
sealing said well, into the well. A continuous flow of culture
medium through the well is then provided by supplying the culture
medium through the inlet conduit and by removing culture medium
through the outlet conduit.
[0168] In an embodiment wherein a system comprising one or more
wells being in fluid communication with each other, a continuous
exchange of culture medium can be provided in that fresh culture
medium is supplied through the inlet opening of the plate sealing
means while being mounted to the corresponding multiwell plate, and
removing culture medium through the outlet opening of the plate
sealing means.
[0169] In an additional and/or alternative embodiment, the flow of
culture medium is generated by utilizing hydrostatic forces. For
example in that the level of culture medium in/above the inlet
opening is kept higher than in/above the outlet opening. In an
alternative embodiment, a flow of culture medium through the wells
being in fluid communication with each other can be achieved in
that the system is placed on a rocker and seesawing the system.
[0170] According to the third aspect, the invention provides the
use of the system as described herein before for propagating cells,
preferably eukaryotic cell, more preferably mammalian cells, and
particularly preferably human cells, most preferably in form of a
microtissue or spheroid.
[0171] In an embodiment of the third aspect, the use of the system
for propagating cells and/or microtissues comprises the use of the
system for shipping cells and/or microtissues. The system for
propagating cells and/or microtissues is particularly advantageous
for shipping cells and/or microtissues due to the tight sealing of
the wells of the multiwell plate by the corresponding plate sealing
means and its properties. The use of the system for shipping cells
and/or microtissues provides better survival rates and better
viability of the cells and/or microtissues at their destination.
Particularly advantageous is that the cells and/or microtissues can
be shipped at a temperature of between 18.degree. C. and 27.degree.
C., preferably at a temperature of between 21.degree. C. and
25.degree. C., and that it is not necessary to ship the cells
and/or microtissues in frozen or deep frozen conditions for
maintaining their survival and viability at a desirable level.
[0172] According to further aspects, the invention provides
multiwell plates as described herein above as part of the system
according to the first aspect, and plate sealing means as described
herein above as part of the system according to the first
aspect.
* * * * *