U.S. patent application number 15/006870 was filed with the patent office on 2016-05-19 for clamping insert for cell culture.
The applicant listed for this patent is CSEM Centre Suisse d'Electronique et de Microtechnique SA Recherche et Developpement. Invention is credited to Silvia ANGELONI SUTER, Martha LILEY.
Application Number | 20160137964 15/006870 |
Document ID | / |
Family ID | 44587681 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160137964 |
Kind Code |
A1 |
ANGELONI SUTER; Silvia ; et
al. |
May 19, 2016 |
CLAMPING INSERT FOR CELL CULTURE
Abstract
A clamping insert for cell culture having a lower support with a
hollow member engagable in a well of a microplate, the hollow
member being open at both ends and including a support surface at
one end, and first tightening elements. The clamping insert further
having an inner holder sized to be fitted inside the hollow member,
and second tightening elements arranged to cooperate with the first
tightening elements of the hollow member to tighten reversibly
between the inner holder and the support surface of the lower
support a porous substrate. The inner holder further including an
open channel keeping free the porous substrate. Retaining elements
intended to cooperate with the well of the microplate are provided
to allow the clamping insert to be suspended in the well, and
sealing elements are provided to hermetically seal the contact area
between the clamping insert and the porous substrate.
Inventors: |
ANGELONI SUTER; Silvia;
(Saint-Blaise, CH) ; LILEY; Martha; (Saint-Blaise,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSEM Centre Suisse d'Electronique et de Microtechnique SA Recherche
et Developpement |
Neuchatel |
|
CH |
|
|
Family ID: |
44587681 |
Appl. No.: |
15/006870 |
Filed: |
January 26, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13554370 |
Jul 20, 2012 |
|
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|
15006870 |
|
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Current U.S.
Class: |
435/287.1 ;
29/559; 422/560 |
Current CPC
Class: |
C12M 23/00 20130101;
C12M 25/08 20130101; Y10T 29/49998 20150115; C12M 41/46 20130101;
C12M 25/04 20130101; C12M 23/48 20130101 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 2011 |
EP |
11174937.0 |
Claims
1. A clamping insert for cell culture comprising: a lower support
comprising: i. a hollow member sized to be engaged in a well of a
microplate, said hollow member having an upper end with a first
opening, a lower end with a second opening, an interior wall
extending between the first and second openings and defining a open
channel therebetween, and a support surface at lower end, said
support surface extending inwardly from the interior wall and
including a central opening so as to defining an open passage
between an uppermost surface of the hollow member and a lowermost
surface of the hollow member, and ii. a first tightening element,
and iii. a retaining element comprising flanges extending, at the
upper end, outwardly from an outside wall of the hollow member
oppositely to the lower end comprising said support surface,
wherein said retaining element is adapted to cooperate with an
upper surface of the well of the microplate to allow the hollow
member a) to be suspended in the well of the microplate, via a
lower surface of the retaining element resting on the upper surface
of the well of the microplate, and b) to divide the well of the
microplate into an apical compartment and a basolateral compartment
in communication via a porous substrate; an inner holder sized to
be fitted inside the open channel of the hollow member, and
comprising a lowermost surface and a second tightening element that
cooperates with said first tightening element of the hollow member
to clamp reversibly said porous substrate between the lowermost
surface of the inner holder and the support surface of the lower
support, said inner holder comprising an open channel extending
from an uppermost surface of the inner holder to the lowermost
surface of the inner holder and arranged to provide access to the
porous substrate; wherein the inner holder is situated entirely
within the open channel of the hollow member; and a seal, that when
placed in contact with said porous substrate, hermetically seals a
contact area between said porous substrate and one of a group
consisting of the inner holder and the lower support.
2. The clamping insert of claim 1, wherein an inner wall of the
inner holder also comprises a guiding groove oriented
longitudinally, allowing passage of a pipette or of a TEER (Trans
Epithelial Electrical Resistance) electrode.
3. The clamping insert of claim 1, wherein said seal is interposed
between the inner holder and the porous substrate and/or interposed
between the porous substrate and the lower support.
4. The clamping insert of claim 1, wherein the inner holder and/or
the lower support is/are made of PEEK, polycarbonate, polystyrene
or other plastics compatible with cell culture.
5. An assembly of a clamping insert for cell culture comprising: a
lower support comprising: i. a hollow member sized to be engaged in
a well of a microplate, said hollow member having an upper end with
a first opening, a lower end with a second opening, an interior
wall extending between the first and second openings and defining a
open channel therebetween, and a support surface at lower end, said
support surface extending inwardly from the interior wall and
including a central opening so as to defining an open passage
between an uppermost surface of the hollow member and a lowermost
surface of the hollow member, and ii. a first tightening element,
and iii. a retaining element comprising flanges extending, at the
upper end, outwardly from an outside wall of the hollow member
oppositely to the lower end comprising said support surface,
wherein said retaining element is adapted to cooperate with an
upper surface of the well of the microplate to allow the hollow
member a) to be suspended in the well of the microplate, via a
lower surface of the retaining element resting on the upper surface
of the well of the microplate, and b) to divide the well of the
microplate into an apical compartment and a basolateral compartment
in communication via a porous substrate; an inner holder sized to
be fitted inside the open channel of the hollow member, and
comprising a lowermost surface and a second tightening element that
cooperates with said first tightening element of the hollow member
to clamp reversibly said porous substrate between the lowermost
surface of the inner holder and the support surface of the lower
support, said inner holder comprising an open channel extending
from an uppermost surface of the inner holder to the lowermost
surface of the inner holder and arranged to provide access to the
porous substrate; wherein the inner holder is situated entirely
within the open channel of the hollow member; a seal, that when
placed in contact with said porous substrate, hermetically seals a
contact area between said porous substrate and one of a group
consisting of the inner holder and the lower support; and a porous
substrate clamped between the lower support and the inner holder,
so that the porous substrate can be removed from the insert only by
untightening and disassembling the inner holder and the lower
support.
6. The assembly of claim 5, wherein the porous substrate is made of
Si3N4.
7. The assembly of claim 5, wherein the porous substrate is micro
machined.
8. The assembly of claim 5, wherein the porous substrate comprises
a silicon nitride membrane supported on a silicon frame.
9. The assembly of claim 5, integrating pads and electrical
circuits for electrical measurements, wherein the substrate is
provided with integrated electrodes and in that the insert is
equipped with contact pads designed to be in contact with the
electrodes.
10. A method for clamping a porous substrate with an insert as
claimed in claim 1, comprising the following steps: unfitting the
inner holder and the lower support, positioning said porous
substrate on the support surface of the lower support, positioning
the inner holder so as to clamp the porous substrate between the
inner holder and the lower support, and tightening the inner holder
with the lower support utilising a dedicated tool comprising some
driving structures intended to cooperate with corresponding
structures arranged in the inner holder.
Description
DESCRIPTION TECHNICAL FIELD
[0001] The present invention relates to the field of cell biology
and cell culture. It more particularly relates to inserts used
together with microwell plates to perform various biological
experiments.
STATE OF THE ART
[0002] In vitro models of biological barriers (such as lung, skin,
the intestines and the blood-brain barrier) are used, for example,
to screen potential pharmaceuticals and toxins for their ability to
enter into and to move around the human body. In vitro models
consist of a single layer or multiple layers of cells that are
cultured in the laboratory so as to mimic the properties of
biological barriers in the body.
[0003] The function of biological barriers in the body is to divide
the inside of the body from the outside (e.g skin, lungs, the
intestines) or different compartments of the body from each other
(e.g. the blood-brain barrier, the walls of blood vessels). In
order to model this function in the laboratory, in vitro models of
biological barriers are cultured on porous membranes. This is
commonly done using microporous well inserts.
[0004] The well insert is used in combination with a microwell
plate consisting of a number of wells made in plastic. The insert
is inserted into a well, which it divides in two, a top (apical)
compartment and a bottom (basolateral) compartment which
communicate via a porous membrane at the bottom of the insert.
Cells are added to the apical side of the well insert and are
cultured on the porous membrane. Typically, the cells will grow to
form a watertight layer that divides the apical from the
basolateral compartment, as in the body.
[0005] The porous membrane is usually made of polymers or of
inorganic aluminium oxide. It is welded, moulded or glued to the
wall of the insert, so as to obtain a perfect seal. Indeed, it is
necessary that the exchanges of material between apical and
basolateral compartments take place only through the microporous
membrane and the cell layer upon it. The membrane and the insert
form a disposable assembly.
[0006] To study, for example, pharmaceutical drug transport or
permeation across the model biological barrier, candidate drugs are
added to the apical compartment, which represents the outside of
the body for a skin model, or the inside of the lungs, or of the
intestines, etc. . . . Permeation is quantified by measuring the
concentration of the candidate drug in the basolateral compartment,
which represents the inside of the body, after a fixed time.
[0007] Recently, a new type of porous substrate has been developed
using microfabrication technology. Such substrates are fabricated
by first depositing a thin layer of ceramic material, such as
Si.sub.3N.sub.4, on a silicon wafer. Pores are then etched in the
Si.sub.3N.sub.4 by photolithography followed by a dry etch. The
silicon wafer is then etched from the other side to remove the
entire thickness of silicon in selected areas, leaving a set of
supports for the transparent porous substrate that remain after
removal of the silicon. The resulting porous substrate comprises a
silicon nitride membrane supported on a silicon frame which gives
it suitable mechanical properties. Thus, this kind of substrate is
rigid.
[0008] Some advantageous of this type of porous substrates in
comparison with other membranes, are listed hereafter: [0009] much
thinner membranes (less than 1 micrometer thick) can be fabricated,
namely at least 20 times thinner than the commonly-used membranes
in commercial well inserts; [0010] pore sizes, shapes, densities
and distributions in the membrane can be tuned as desired; [0011]
the membrane is highly transparent in both air and water
independently of the pore size and density; [0012] the membranes
are resistant to acids, bases, solvents, high temperatures and
e-beam exposure; [0013] the membranes are reusable
(reconditionable) after cell culture.
[0014] These properties are available advantageously in one unique
combination together with the properties commonly exhibited by the
existing membranes such as: [0015] low intrinsic fluorescence of
the membrane; [0016] possible chemical pretreatment to enhance cell
culture [0017] good cell growth in general and, in particular, the
formation of tight layers of epithelial cells; [0018] they
withstand common sterilization procedures.
[0019] However, this kind of rigid substrate presents two major
drawbacks. Firstly, it is more expensive than the commonly used
polymer microporous substrates. This can be overcome by its reuse.
In addition, it requires a specific holder that makes it compatible
with a commercial well plate and also with routine laboratory
practice.
[0020] A cell culture insert has been proposed in US2008076170.
Such insert includes a body having a grip extending sideways from
the body and a locking ring adapted to be fitted around the body.
The cell culture insert is kept in place by friction contact
against interior sidewalls of a cavity of a cell culture vessel.
Its height position can be adjusted inside said cavity.
[0021] However, the insert as disclosed may only be used with a
flexible membrane, able to be folded between the locking ring and
the body. Such an embodiment does not define a tight seal but
allows culture medium to circulate from one side of the membrane to
another without crossing through the membrane, but by gaps
occurring between the membrane and the body or between the membrane
and the locking ring. Therefore, this clamped membrane, because of
these gaps, does not define two reaction chambers with exclusive
exchange of solvent and solutes through the membrane itself.
Therefore the taught insert cannot be used to investigate transport
properties through model biological barriers. Moreover,
conventional sealing means can not be directly implemented, since
it may not be compatible with a sliding engagement of the locking
ring on the body.
[0022] Thus, the present invention aims to alleviate these problems
and allow a practical use of this type of rigid substrate.
SUMMARY OF THE INVENTION
[0023] To this end, the invention concerns a clamping insert for
cell culture comprising: [0024] a lower support comprising: [0025]
i. a hollow member sized to be engaged in a well of a microplate,
said hollow member being open at its both ends and comprising a
support surface at one of its ends, [0026] ii. first tightening
means, [0027] an inner holder sized to be fitted inside the hollow
member, and comprising second tightening means arranged to
cooperate with said first tightening means of the hollow member to
tighten reversibly between the inner holder and the support surface
of the lower support a porous substrate, said inner holder
comprising an open channel keeping free the porous substrate;
[0028] retaining means intended to cooperate with the well of the
microplate to allow the clamping insert to be suspended in the well
of the microplate, [0029] sealing means to hermetically seal the
contact area between the inner holder and said porous
substrate.
[0030] Some other advantageous characteristics are specified in the
claims.
[0031] The invention also concerns an assembly comprising such a
clamping insert and a porous substrate. The invention also concerns
a method for clamping a porous substrate with such an insert. Said
method comprises the following steps: [0032] unfitting the inner
holder and the lower support, [0033] placing said porous substrate
on the support surface of the lower insert, [0034] disposing the
inner holder so as to clamp the porous substrate between the inner
holder and the lower support, [0035] tightening the inner holder in
the lower support.
[0036] A dedicated tool comprising some driving structures intended
to cooperate with corresponding structures arranged in the inner
holder may be used for the tightening step if appropriate.
BRIEF DESCRIPTION OF THE FIGURES
[0037] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention, and, together with the general description of the
invention given above, and the detailed description of the
embodiments given below, serve to explain the principles of the
present invention.
[0038] FIGS. 1a and 1b are two perspective views of an embodiment
of the lower support, FIG. 1c represents an anti-wear ring intended
to cooperate with the lower support.
[0039] FIGS. 2a and 2b are two perspective views of an embodiment
of the inner holder, FIG. 2c represents a seal intended to
cooperate with the inner holder.
[0040] FIG. 3 show the assembled clamping insert according one
embodiment of the invention.
[0041] FIG. 4 represents an example of an assembling/disassembling
tool adapted to the invention.
[0042] FIGS. 5 and 6 propose sketches of two other embodiments of
the invention.
DETAILED DESCRIPTION
[0043] FIGS. 1, 2 and 3 represent a preferred embodiment of an
insert according to the invention. This insert aims to facilitate
the use as a biological substrate of a porous substrate made with a
Si.sub.3N.sub.4 layer deposited on a silicon wafer and then
micromachined. Such a porous substrate 10 can be seen on FIGS. 5
and 6. This substrate as such is not part of the invention and will
not be described in detail. One can refer to Madou, M. J,
Fundamental of Microfabrication: the science of miniaturization,
Second Edition, CRC Press 2002, for more information about this
technology. It should be noted that such substrates are rigid.
[0044] It should be noticed that, due to specifications and costs
of the substrate, it should preferably be arranged in a reusable
way.
[0045] Thus, to this end, the insert according to the invention
comprises a lower support 12 (FIGS. 1a and 1b) comprising a hollow
member 14 sized to be engaged in a well of a microplate, preferably
of a standard microplate. The hollow member 14 could be made in
different suitable materials, like plastic or aluminium. PEEK
(polyether ether ketone), PC (polycarbonate), polystyrene or
similar polymers are well suited since they resist typical
sterilization conditions and they can be injection moulded in a
low-cost production process.
[0046] The hollow member 14 is open at both its ends. At one end,
its wall defines a reverse side which forms a support surface 16
for the porous substrate 10. The lower support 12 furthermore
comprises some flanges 18 extending from the wall of the hollow
member 14 oppositely to the end comprising said support surface 16.
These flanges 18 define retaining means. They are sized and
arranged so as to allow the lower support 12 and the whole insert
to rest on the sides of the microplate well and so as to avoid the
whole assembly touching the bottom of the microplate well.
[0047] The lower support 12 furthermore comprises first tightening
means. In the detailed example, these first tightening means take
shape of two grooves 20, diametrically opposed. Each groove 20
comprises a first longitudinal part, parallel to the longitudinal
axis of the cylinder of the hollow member 14. This first
longitudinal part is extended by a second inclined part,
essentially perpendicular to the longitudinal axis but with a
slight slope directed toward the support surface 16.
[0048] The insert according to the invention comprises furthermore
an inner holder 22 (FIGS. 2a and 2b) sized to be adjusted inside
the hollow member 14. More precisely, the inner holder 22 is
similar to the hollow member 14, the exterior dimension of which
being slightly lower than the interior dimensions of the hollow
member 14. In this example, the inner holder 22 has a cylindrical
shape. This inner holder 22 can be made of plastic, especially PEEK
(polyether ether ketone), PC (polycarbonate), polystyrene or the
like.
[0049] The inner holder 22 comprises second tightening means
arranged to cooperate with the first tightening means. In this
example, said second tightening means are two pins 24 diametrically
opposed and sized to be engaged in the grooves 20 of the hollow
member 14, so as to form a bayonet fitting. The pins 24 and grooves
20 could also be disposed reversely. A torque limiting system could
be implemented so as to avoid damaging the porous substrate.
[0050] The length of the inner holder 22 is sized so that, when the
inner holder 22 is tightened on the lower support 12, a porous
substrate 10 may be clamped between both of them.
[0051] Thus, there may remain a free space between the support
surface 16 and the end of the inner holder 22, with a height lower
than the thickness of the porous substrate. When present, the
porous substrate is held firmly in this free space. To decrease the
pressure and/or improve the clamping, interfaces can be fitted with
suitable coatings in rubber, Teflon or other elastic materials
which will also reduce wear.
[0052] As explained above, it is of utmost importance that apical
and basolateral compartments be well separated so that exchanges
between them be only implemented through the substrate and the
cells grown on it.
[0053] For this purpose and according an advantageous aspect of the
invention, the insert includes a sealing means to hermetically seal
the contact area between the inner holder 22 and said porous
substrate. One could also consider disposing the seal 26 between
the substrate end the lower support 12. FIGS. 5 and 6 show that it
is possible to dispose two seals, one on each side of the porous
substrate 10.
[0054] For example, sealing means may be implemented with a
silicone ring 26 (FIG. 2c) interposed between the inner holder 22
and the porous substrate 10. Other suitable materials may be used
for the seal 26, like rubber or elastic polymers.
[0055] An annular recess may be also engraved in the substrate, in
which the seal may take place. Thus, the seal 26 is perfectly
positioned and the tightness area is perfectly defined around the
porous membrane and the culture zone.
[0056] The insert according to the invention may also present some
interesting and advantageous features.
[0057] For instance, the lower support 12 may comprise grooves 28
to allow easy release of air bubbles from below the porous
membrane.
[0058] The inner wall of the inner holder 22 may also comprise a
guiding groove 30 oriented longitudinally, for both pipetting and
TEER (Trans Epithelial Electrical Resistance) electrodes. The
guiding groove 30 may guide the tip of a pipette up to 1 mm above
the porous substrate 10 without touching the cell culture and
membrane damaging. The same groove 30 can be used to easily
position reproducibly the electrodes used to record TEER
values.
[0059] As mentioned above, the support surface 16 of the lower
support 12 may be equipped with some antiwear means. For example,
it may be coated with a suitable antiwear coating or comprise an
antiwear ring 31 (FIG. 1c). Teflon or other low friction polymers
are well suited as antiwear means.
[0060] To enhance the use of the insert according to the invention,
FIG. 4 also proposes a tool 32 designed to assemble/disassemble
easily the inner holder 22 on the lower support 12, reducing any
risk of contamination or of wrong manipulation. Such tool 32
comprises a grip to be handled by a user or by a robot. Said tool
32 also comprises some driving structures 34 able to cooperate with
corresponding structures arranged in the inner holder 22. Once the
driving structures 34 are engaged with the corresponding structures
of the inner holder 22, one can tighten/untighten the inner holder
22 in the lower support 12 by rotating the tool 32 in one or in the
other direction, while the lower support 12 is fixed. As shown in
FIG. 4, the driving structure may be obtained by screws or pins,
while the corresponding structure may be obtained by grooves
realized on the top end of the inner holder 22. One can implement a
torque limiting system in the tool grip or between the grip and the
driving structure, to avoid damaging the substrate.
[0061] Other tightening means could also be considered. For
example, FIG. 5 proposes to assembly the inner holder 22 by
clipping it on the lower support 12. To this end, the inner holder
22 may comprise some elastic extensions 40, sized and shaped to
hook a rim 42 or the flanges of the lower support 12. The elastic
properties of the extensions allow, when the inner holder is
hooked, to fix rigidly these two parts and to clamp the porous
substrate in between. The elastic properties allow however to
unclip these two parts.
[0062] FIG. 6 proposes to assembly the inner holder 22 by screwing
it on the lower support 12. Similarly to the embodiment of FIG. 5,
the inner holder 22 may comprise some extensions 40, comprising
first screwing means 44 (i.e. thread or tapping), sized and shaped
to cooperate with second screwing means 46 (respectively tapping or
thread) of a rim 42 of the lower support 12. These two parts can be
screwed in order to clamp the porous substrate in between.
[0063] Thus, the insert according to the invention allows clamping
a porous substrate 10 comprising a silicon nitride membrane
supported on a silicon frame and obtained by microfabrication, in
an efficient and practical way. The holder is key to the use of the
porous substrate, the design of which can be tuned according to the
most precise requirements of the cell culture. One can therefore
benefit from the advantages of such a substrate, as detailed in the
introduction. Indeed, despite the very tight seal, the substrate
can easily be removed after an experiment and can be reconditioned,
i.e. cleaned to obtain a bare porous substrate 10 and reused for a
subsequent cell culture. Furthermore, the assembling and
disassembling procedure allows the membrane to be turned upside
down and still incubated with the physiological buffer. These
properties may improve the co-culture procedure. As explained
above, the insert is well suited for TEER measurements.
[0064] In a further improvement of the insert according to the
invention, one could consider integrating the relevant contact pads
and circuits for TEER measurements. For example, thanks to its
silicon basis, the substrate could be provided with integrated
electrodes. The insert could also be equipped with contact pads
designed to facilitate electrical contact with the external
equipment. This would greatly improve the reproducibility of
electrical measurements.
[0065] In another improvement of the insert, one could consider
integrating a wave guide in the inner holder 22, such wave guide
being used to bring the light close the surface bearing the culture
cell, either for growth improvement or the illumination for optical
inspection or for preculture UV sterilization.
[0066] The examples above should not be considered as limiting.
Those skilled in the art will appreciate that numerous
modifications can be made thereof without departing from its
spirit. The clamping insert according to the invention can be used
for any kind of rigid substrates. The scope of the invention is to
be determined by the appended claims and their equivalent.
* * * * *