Insert for inserting into a growth medium, method for the same

Abstract

The invention relates to an insert for inserting into a cell culture vessel. The invention further relates to an insert assembly comprising multiple inserts according to the invention. The invention further relates to a lid for a cell culture vessel comprising an insert according to the invention. The invention further relates to an assembly for cell growth comprising a cell culture vessel, and an insert, insert assembly or lid according to the invention. The invention further relates to a method for cell growth wherein use is made of an insert according to the invention.

Description

[0001] WU R24002WG / P36805PC00 - 1 -

[0002] INSERT FOR INSERTING INTO A GROWTH MEDIUM, METHOD FOR THE SAME

[0003] The invention relates to an insert for inserting into a growth medium, for example contained in a cell culture vessel. The invention further relates to an insert assembly comprising multiple inserts according to the invention. The invention further relates to a lid for a cell culture vessel comprising an insert according to the invention. The invention further relates to an assembly for cell growth comprising a cell culture vessel, and an insert, insert assembly or lid according to the invention. The invention further relates to a method for cell growth wherein use is made of an insert according to the invention.

[0004] In the art, cell culture vessels, such as flasks, tubes or wells in well plates are used to grow cells. A cell growth medium is provided in the cell culture vessels which medium may provide the cells with the nutrition needed to grow. A problem associated herewith is that hypoxia in the cell cultures may occur due to a lack of sufficient oxygen. For example, oxygen may not reach cells that are present deeper in the cell growth medium such that only cells on an outside of the cell growth medium or cell culture vessel receive sufficient oxygen to grow.

[0005] In the art, oxygen may be provided to the cells in the cell growth medium by using direct bubbling. However, a downside of this approach is that the cells in the cell growth medium may be damaged by the bubbles, that foaming problems may arise and / or that problematic hydrodynamic and shear stress occur on the cells.

[0006] Further, it is known that the growth of plants and other organisms in a growth medium, such as soil or a water-based substrate, is influenced by various factors, including the availability of oxygen in the growth medium. Insufficient oxygen levels can negatively affect growth and development. However, maintaining adequate oxygen levels in the growth medium presents a significant challenge. Existing methods, such as introducing tubing into the soil to deliver oxygen directly, have limitations, including uneven oxygen distribution and potential clogging. Accordingly, there remains a need for improved methods and systems to enhance oxygen availability in growth media effectively and efficiently.

[0007] The invention aims to address the challenge of providing an adequate supply of gas, such as oxygen, to the growth medium to allow for growth of cells and / or organisms grown therein. A further aim of the invention is to mitigate hypoxia in cells within cell culture vessels, such as well plates. A further aim of an aspect of the invention is to improve oxygen supply in agriculture applications such as water-logged soils, hydroponic systems or other plant growth systems. WU R24002WO / P36805PC00 - 2 -

[0008] The aims of the invention are achieved by an insert according to claim 1.

[0009] The insert of the invention is configured for inserting into a growth medium.

[0010] The growth medium may comprise, or be, soil for cultivating organisms, e.g. plants or fungi, therein. The growth medium may be present in a green house or an open field. The insert may have dimensions determined based on a specific organism grown in the growth medium and / or an ability of the gas, e.g. oxygen, to move through the growth medium from the insert to the organism. For example, when growing a plant with an extensive root system, the insert may be larger compared to when growing a fungus with a more localized mycelial distribution.

[0011] The growth medium may also be a growth medium, e.g. a liquid growth medium, contained in a cell culture vessel, e.g. to grow cells. An example of a cell culture vessel is a well of a well plate. The cell culture vessel may contain a cell growth medium. The cell culture vessel may comprise a circumferential side wall, a bottom wall, and an open top side to allow access to the cell growth medium contained in the cell culture vessel. In embodiments, the cell culture vessel forms an intrusion in a plate, e.g. the cell culture vessel comprising a sloping bottom wall, such that the cell growth medium may be contained in the cell culture vessel.

[0012] The insert is configured to be inserted into the growth medium, e.g. into the cell culture vessel which contains the growth medium.

[0013] In embodiments, the insert may have an elongated shape such that it may be provided in the growth medium, e.g. the cell, parallel to a line of organisms grown therein. For example, the elongated insert may be provided adjacent and parallel to a line of plants grown in the soil. In embodiments, lines of plants are alternated with elongated inserts such that the inserts provide gas to adjacent lines of plants.

[0014] In embodiments, the insert may have a dimension, e.g. a height, that is slightly smaller than a distance between a top side of the cell culture vessel and a bottom side thereof so that the insert does not come into contact with the bottom side of the cell culture vessel allowing a space, i.e. an opening, between the bottom side of the cell culture vessel and a bottom side of the insert when the insert is inserted into the cell culture vessel. In principle, the insert may have any shape such that it does not interfere with the interaction of the cells and the culture vessel surfaces, while providing sufficient surface area for gas transfer. WU R24002WG / P36805PC00 - 3 -

[0015] In embodiments, the cell growth medium contained in the cell culture vessel may be any type of liquid medium used to culture cells, such as DMEM, RMPI or other media, with or without the addition of serum or other nutrients or antibiotics.

[0016] The insert comprises an inner space therein, which inner space allows contains a liquid gas-carrier. An example of a liquid gas-carrier is a material comprising of a fluorocarbon. Due to that the inner space is separated from an outside ambient, the growth medium will not come into direct contact with the gas carrier contained in the insert. The gas carrier may carry a gas to improve growth in the growth medium, e.g. the gas may be oxygen or a gas mix comprising oxygen.

[0017] The inner space is at least partly defined by a gas-permeable membrane separating the inner space from the growth medium when the insert is inserted into the growth medium. The membrane is configured for allowing a gas, e.g. oxygen, carried by the gas carrier in the inner space to move, e.g. diffuse, from the gas carrier in the inner space through the membrane to the growth medium when the insert is inserted into the growth medium. Preferably, the gas- permeable membrane does not block cell adherence in the culture vessel, where relevant.

[0018] Preferably, the gas-permeable membrane is manufactured from a gas-porous material such as a silicone material.

[0019] The gas-permeable membrane, which may be manufactured from a gas-porous material, has a varying (non-uniform) thickness along a given direction, e.g. a vertical or height direction, such as a direction parallel to a side wall of a well when the insert is placed therein. The varying thickness allows for a spatially varying gas-flux profile through the membrane. Consequently, designated areas of the growth medium may receive a higher gas influx, while other areas receive less gas. Depending on the specific thickness profile, for example a parabolic or sinusoidal variation, this may further give rise to a circulation pattern of gas within the growth medium. Gas may enter the medium through a locally thinner portion of the membrane and re-enter the inner space through another thinner portion of the same membrane or of a different membrane .

[0020] An advantage of the insert of the invention is that the insert may be readily inserted and removed from the growth medium causing minimal disruptions in the cultures contained therein. In preferred embodiments, the cells themselves will not be in direct contact with the insert, as the region of the culture vessel where the cells rest or attach (typically the bottom of the plate or flask), is not covered by the insert. WU R24002WO / P36805PC00 - 4 -

[0021] Furthermore, by suitably changing dimensions and shapes of the insert, sufficient gas, e.g. oxygen, may be provided to the cultures in the growth medium by the insert of the invention. For example, the amount of oxygen needed, and location in the growth medium where the oxygen is needed, may depend on the type of organism and / or cells grown. For example, it may depend on a complexity of the cell culture grown, e.g. 3D cell cultures, and size of the cell culture. For example, an amount of contact surface between the insert and the growth medium may depend on a shape and size of the insert. The surface-area-to-volume ratio of the insert may be selected to adjust the rate of oxygen delivery to the growth medium. By choosing an insert with a suitable ratio, the oxygen supply can be tuned to the requirements of a given application. In addition, oxygen delivery may further be controlled by selecting an insert having a specific varying thickness profile of the gas-permeable membrane.

[0022] Preferably, the insert delivers gas to the medium solely by passive diffusion, typically without any gas inlet, gas outlet, gas-flow channel, bubbling, or other form of active gas flow being in contact with the cell growth medium.

[0023] In embodiments, the insert may have a substantially ring shape, i.e. having an annular crosssection, such that, when the insert is provided into the growth medium, the growth medium may be accessed via the central opening of the ring shape, e.g. via a top side thereof.

[0024] The amount of gas exchange between the inner space and the cell culture or organism may further be controlled by adapting a volume of the gas carrier, a size of the membrane, an addition of a further gas mixture to the inner space, a flow of the gas carrier, a thickness of openings in the membrane. The invention may prevent flowing gas from coming into direct contact with the cell medium. This avoids bubbling or foaming in the cell growth medium.

[0025] As a result of the gas moving through the membrane, no bubbling of the gas is needed avoiding the downsides associated therewith.

[0026] In embodiments, the liquid gas-carrier in the inner space remains substantially static during use, typically with no bulk flow other than passive diffusion of gas.

[0027] In embodiments, the insert comprises an insert handle on a top side thereof, e.g. wherein the top side of the insert is opposite the bottom side of the insert. The insert handle may be used to handle the insert, e.g. for inserting and / or removing the insert from the growth medium without contacting the portion of the insert inside the growth medium. WU R24002WO / P36805PC00 - 5 -

[0028] In embodiments, the inner space further comprises a gas mixture, preferably wherein the gas mixture depends on the cell growth in the cell growth medium.

[0029] In embodiments, the insert is manufactured from a polymer material, such as a synthetic polymer material, preferably wherein the material is heat-stable and / or biologically inert. The insert may be manufactured from one or more of the following materials:

[0030] - polysiloxanes;

[0031] - silicone oil;

[0032] - fluorinated polysiloxanes;

[0033] - fluorinated hydrocarbons;

[0034] - polyethers;

[0035] - polyperfluoroalkyl oxides;

[0036] - polytetrafluoroethylene; and

[0037] - polyethylene.

[0038] Preferably, the insert is manufactured such that cells do not stick thereto, e.g. the insert may be manufactured from a biologically inert material.

[0039] In embodiments, the growth medium is a cell growth medium contained in a cell culture vessel such as a well in a well plate.

[0040] In embodiments, the insert comprises one or more insert supports, which one or more insert supports protrude from a bottom side of the insert for supporting the insert on a wall of the cell culture vessel when inserted therein such that a cell growth space is present between the bottom side of the insert and a bottom wall of the cell culture vessel.

[0041] In embodiments, an outer shape of the insert substantially matches a shape of a crosssection of the cell culture vessel, and / or wherein a size of the insert substantially matches a size of the cell culture vessel.

[0042] In embodiments, the insert has an annular cross-section such that, when the insert is inserted into the cell culture vessel, the cell growth medium in the cell culture vessel is accessible through an opening of the annular cross-section.

[0043] In embodiments, the insert further comprises a gas carrier control system connected to the inner space of the insert, for example wherein the gas carrier control system comprises a processor and / or a gas carrier pump, which gas carrier control system, e.g. the processor and / or pump thereof, is configured to: WU R24002WO / P36805PC00 - 6 -

[0044] - provide a flow of the gas carrier in the inner space;

[0045] - add to and / or remove from the inner space a predetermined amount of gas carrier; and / or

[0046] - add to and / or remove from the inner space a predetermined amount of gas from the gas carrier.

[0047] For example, in these embodiments the inner space of the insert may be functionally connected to the gas carrier control system via a tubing, e.g. a flexible tubing, such that the gas carrier control system may affect the gas carrier contained in the inner space. For example, the connection between the control system and the inner space may be separable such that the insert, e.g. the portion not comprising the gas carrier control system, may be sterilized and / or renewed away from the gas carrier control system.

[0048] In embodiments, the growth medium comprises a typically porous plant-cultivation medium, including but not limited to soil, water-logged soil, or a hydroponic substrate, and the insert is configured to deliver gas by passive diffusion into said medium when placed therein.

[0049] The invention further relates to an insert assembly comprising multiple inserts, e.g. comprising an appropriate number to fit in chambers of a culture vessel (e.g. 6 inserts for a 6- well plate), according to the invention, wherein the inserts are rigidly provided in a predetermined pattern, e.g. depending on a distribution of associated cell culture vessels, e.g. a distribution of wells in a well plate. In this way, multiple inserts of the invention may be inserted or removed from multiple cell culture vessels allowing for a more efficient use thereof. The inserts of the insert assembly may differ, e.g. have different sizes and / or shapes, and contain different gas carriers and / or gasses in the inner spaces thereof.

[0050] In embodiments, the inner spaces of the inserts, e.g. of at least some of the inserts, of the insert assembly are connected, e.g. via hollow insert connectors, allowing the gas carrier to flow between the inner spaces. This may allow for example the gas carrier control system to add and / or remove gas carrier from multiple inserts of the insert assembly by creating one gas carrier flow.

[0051] The invention further relates to a lid for a cell culture vessel, e.g. for a well of a well plate, comprising an insert according to the invention or an insert assembly according to the invention. The lid may be used to cover multiple cell culture vessels, the lid being provided with multiple inserts according to the invention, each insert having an associated cell culture vessel, wherein the inserts are provided on the lid in a pattern corresponding to a pattern of the associated cell culture vessels. WU R24002WO / P36805PC00 - 7 -

[0052] The lid may allow for a stack of cell culture vessels, wherein a second cell culture vessel may be stacked on top of a lid provided on a first cell culture vessel.

[0053] The invention further relates to an assembly for cell growth comprising:

[0054] - a cell culture vessel, e.g. a well of a well plate, for containing a cell growth medium therein, wherein the cell culture vessel comprises a side wall surrounding the cell culture vessel and a bottom wall displaced from a top side of the cell culture vessel by the side wall; and

[0055] - an insert according to the invention, an insert assembly according to the invention, or a lid according to the invention, wherein the corresponding insert, when inserted into the cell culture vessel, at least partially extends from the top surface to the bottom wall of the cell culture vessel.

[0056] The insert, insert assembly, lid and assembly for cell growth of the invention and the gas carrier contained therein may be re-usable for use in different, e.g. subsequent, cell culture vessels. The insert, insert assembly, lid or assembly for cell growth may have to be cleaned and / or prepared before re-use.

[0057] The invention further relates to a method for cell growth, wherein use is made of an insert according to the invention, an insert assembly according to the invention, a lid according to the invention or an assembly for cell growth according to the invention. The method of the invention may at least comprise the step of inserting the inserts, e.g. of one of the insert assembly, lid, or assembly for cell growth, into a cell culture vessel and allowing gas carried by the gas carrier to move from the inner space of the insert into the cell culture of the cell culture vessel.

[0058] In embodiments, the method comprises:

[0059] - inserting the insert into a cell growth medium in a cell culture vessel.

[0060] In embodiments, the method comprises:

[0061] - providing a flow of the gas carrier in the inner space;

[0062] - adding to and / or removing from the inner space a predetermined amount of gas carrier; and / or

[0063] - adding to and / or removing from the inner space a predetermined amount of gas from the gas carrier.

[0064] Preferably use is made of an embodiment of the invention comprising the gas carrier control system. WU R24002WO / P36805PC00 - 8 -

[0065] The method may further comprise:

[0066] - sterilizing the insert, e.g. using autoclaving.

[0067] For example, the insert may be sterilized N times before the insert may be recycled and / or a new gas carrier carrying a gas has to be provided into the inner space thereof.

[0068] The method may further comprise removing, sterilizing and reusing the insert.

[0069] The invention will be explained below with reference to the drawing in which:

[0070] Fig. 1a shows a first embodiment of an insert;

[0071] Fig. 1 b shows a second embodiment of an insert; and

[0072] Fig. 2 shows an insert assembly with a gas carrier control system.

[0073] Figure 1a shows a first embodiment of an insert 1 for inserting into a growth medium, e.g. contained in a well of a well plate. The insert comprises an inner space 2 containing a gas carrier (indicated by the sloped lines). The gas carrier may carry a gas, such as oxygen, to allow for growth in the growth medium.

[0074] The gas carrier shown in figure 1a does not fully fill the inner space 2, e.g. allowing for a gas mixture, which may not be carried by the gas carrier, to be present above the gas carrier in the inner space 2. The shown insert has a ring shape with an angular cross-section allow access to a cell culture through the opening in a top side of the insert 1.

[0075] The insert 1 further comprises a gas-permeable membrane 3. The membrane 3 separates the inner space 2 from the growth medium when the insert 1 is inserted into the growth medium. The membrane 3 is configured for allowing a gas, e.g. oxygen, carried by the gas carrier in the inner space 2 to move, e.g. diffuse, from the gas carrier in the inner space 2 through the membrane 3 to the growth medium when the insert 1 is inserted into the growth medium. In the embodiment shown, the membrane 3 extends along an inner part of a lower portion of the insert 3. The size and shape of the membrane 3 may depend on the application, e.g. the cell to be grown, of the insert 1.

[0076] The figures do not depict the varying thickness of the gas-permeable membrane 3 due to their schematic nature. However, the embodiments illustrated do include gas-permeable membranes having a variable thickness along a direction thereof so as to provide a spatially varying gas-flux profile. WU R24002WO / P36805PC00 - 9 -

[0077] Figure 1b shows a second embodiment of an insert 1 similar to the insert 1 shown in figure 1a. The insert 1 of figure 1b has a larger height and cross section compared to the insert 1 shown in figure 1a. Further, the inner space 2 of the insert 1 of figure 1b is fully filled with the gas carrier and the membrane 3 of the insert 1 of figure 1 b extends further along an inner side of the insert 1 of figure 1b compared to the membrane 3 shown in figure 1a.

[0078] The size and shape of the inserts 1 may vary. For example, a thickness of the walls of the insert may vary, a size of the membrane may vary, and a gas contained in the gas carrier may vary.

[0079] Figure 2 shows an insert assembly 4 comprising six inserts 1 and a gas carrier control system 5. The gas carrier control system 5 allows for a flow of gas carrier through the inserts 1 that are interconnected via the insert connectors 6, e.g. embodied as hollow tubing connecting the various inner spaces 2 of the inserts 1. By a force applied by the gas carrier control system 5, e.g. using a pump, the gas carrier flow may be caused. This may allow for better distribution of the gas carried by the gas carrier. This may further allow to add more gas to the gas carrier and distributing the gas through the inner spaces 2 efficiently.

[0080] The insert assembly 4 as shown in figure 2 may be part of a lid which may cover, for example, a well plate having various wells wherein the inserts 1 of the insert assembly 4 may be provided when the lid is placed on the well plate.

[0081] Aspects of the invention will be described in the below clauses, in which:

[0082] Clause 1. Insert for inserting into a growth medium, wherein the insert comprises an inner space at least partly defined by a gas-permeable membrane, wherein the gas-permeable membrane separates the inner space from the cell growth medium when the insert is inserted into the growth medium, wherein the gas-permeable membrane is configured for allowing a gas carried by a gas carrier contained in the inner space to move from the gas carrier in the inner space through the gas-permeable membrane to the growth medium when the insert is inserted into the growth medium.

[0083] Clause 2. Insert according to clause 1 , wherein the gas carrier comprises a gas- permeable liquid, such as a fluorocarbon. WU R24002WO / P36805PC00 - 10 -

[0084] Clause 3. Insert according to one or more of the preceding clauses, wherein the insert is manufactured from a polymer material, such as a synthetic polymer material, preferably wherein the material is heat-stable and / or biologically inert.

[0085] Clause 4. Insert according to one or more of the preceding clauses, further comprising a gas carrier control system connected to the inner space of the insert, wherein preferably the gas carrier control system comprises a processor and / or a pump, which gas carrier control system, e.g. the processor and / or pump thereof, is configured to:

[0086] - provide a flow of the gas carrier in the inner space;

[0087] - add to and / or remove from the inner space a predetermined amount of the gas carrier; and / or

[0088] - add to and / or remove from the inner space a predetermined amount of gas from the gas carrier.

[0089] Clause 5. Insert according to one or more of the preceding Clauses, wherein the growth medium is a cell growth medium contained in a cell culture vessel.

[0090] Clause 6. Insert according to clause 5, wherein the insert comprises one or more insert supports, which one or more insert supports protrude from a bottom side of the insert for supporting the insert on a wall of the cell culture vessel comprising the cell growth medium when inserted in the cell growth medium such that a cell growth space is present between the bottom side of the insert and a bottom wall of the cell culture vessel.

[0091] Clause 7. Insert according to one or more of the clauses 5 - 6, wherein an outer shape of the insert substantially matches a shape of a cross-section of the cell culture vessel, and / or wherein a size of the insert substantially matches a size of the cell culture vessel.

[0092] Clause 8. Insert according to one or more of the clauses 5 - 7, wherein the insert has an annular cross-section such that, when the insert is inserted into the cell culture vessel containing the cell growth medium, the cell growth medium is accessible through an opening of the annular cross-section.

[0093] Clause 9. Insert assembly comprising multiple inserts, e.g. comprising at least six inserts, according to one or more of the preceding claims, wherein the inserts are rigidly provided in a predetermined pattern, e.g. depending on a distribution of associated cell culture vessels, e.g. a distribution of wells in a well plate. WU R24002WC / P36805PC00 - 11 -

[0094] Clause 10. Insert assembly according to clause 9, wherein the inner spaces of the multiple inserts are interconnected, e.g. via hollow insert connectors, allowing the gas carrier to flow between the inner spaces.

[0095] Clause 11. Lid for a cell culture vessel, e.g. for a well of a well plate, comprising an insert according to one or more of the clauses 5 - 8 or an insert assembly according to one or more of the clauses 9 - 10.

[0096] Clause 12. Assembly for cell growth comprising:

[0097] - a cell culture vessel, e.g. a well of a well plate, for containing a cell growth medium, wherein the cell culture vessel comprises a side wall surrounding the cell culture vessel and a bottom wall displaced from a top side of the cell culture vessel by the side wall; and

[0098] - an insert according to one or more of clauses 5 - 8, an insert assembly according to clause 9 or 10, or a lid according to clause 11 , wherein the insert, when inserted into the cell culture vessel, at least partially extends from the top side to the bottom wall.

[0099] Clause 13. Method for growth, wherein use is made of an insert according to one or more of the clauses 1 - 8, an insert assembly according to one or more of the clauses 9 - 10, a lid according to clause 11 or an assembly according to clause 12.

[0100] Clause 14. Method according to clause 13, comprising:

[0101] - inserting the insert into a growth medium.

[0102] Clause 15. Method according to clause 13 or 14, comprising:

[0103] - providing a flow of gas carrier in the inner space;

[0104] - adding to and / or removing from the gas carrier in the inner space a predetermined amount of gas carrier; and / or

[0105] - adding to and / or removing from the gas carrier in the inner space a predetermined amount of gas.

Claims

WU R24002WO / P36805PC00 - 12 -CLAIMS1. Insert for inserting into a growth medium, wherein the insert comprises an inner space at least partly defined by a gas-permeable membrane, wherein the gas-permeable membrane separates the inner space from the cell growth medium when the insert is inserted into the growth medium, wherein the inner space contains a liquid gas-carrier, and the gas-permeable membrane is configured to allow a gas carried by the liquid gas-carrier to diffuse from the inner space into the cell growth medium, wherein the gas-permeable membrane has a variable thickness, e.g. an intentional variable thickness, along a direction thereof so as to provide a spatially varying gas-flux profile through the gas-permeable membrane, and wherein the insert is configured to deliver gas from the liquid gas-carrier to the growth medium by passive diffusion, preferably wherein the insert delivers gas to the medium solely by passive diffusion, and wherein the liquid gas-carrier is fully confined within the inner space so that it does not contact the growth medium during use.

2. Insert according to claim 1, wherein the gas carrier comprises a gas-dissolving liquid, such as a fluorocarbon.

3. Insert according to one or more of the preceding claims, wherein the insert is manufactured from a polymer material, such as a synthetic polymer material, preferably wherein the material is heat-stable and / or biologically inert.

4. Insert according to one or more of the preceding claims, further comprising a gas carrier control system connected to the inner space of the insert, wherein preferably the gas carrier control system comprises a processor and / or a pump, which gas carrier control system, e.g. the processor and / or pump thereof, is configured to:- provide a flow of the gas carrier in the inner space;- add to and / or remove from the inner space a predetermined amount of the gas carrier; and / or- add to and / or remove from the inner space a predetermined amount of gas from the gas carrier.

5. Insert according to one or more of the preceding claims, wherein the growth medium is a cell growth medium contained in a cell culture vessel.WU R24002WO / P36805PC00 - 13 -6. Insert according to claim 5, wherein the insert comprises one or more insert supports, which one or more insert supports protrude from a bottom side of the insert for supporting the insert on a wall of the cell culture vessel comprising the cell growth medium when inserted in the cell growth medium such that a cell growth space is present between the bottom side of the insert and a bottom wall of the cell culture vessel.

7. Insert according to one or more of the claims 5 - 6, wherein an outer shape of the insert substantially matches a shape of a cross-section of the cell culture vessel, and / or wherein a size of the insert substantially matches a size of the cell culture vessel.

8. Insert according to one or more of the claims 5 - 7, wherein the insert has an annular cross-section such that, when the insert is inserted into the cell culture vessel containing the cell growth medium, the cell growth medium is accessible through an opening of the annular cross-section.

9. Insert according to one or more of the preceding claims, wherein the cell growth medium comprises a preferably porous plant-cultivation medium, for example soil, waterlogged soil, or a hydroponic substrate, and wherein the insert is configured to deliver gas by passive diffusion into said medium when placed therein.

10. Insert assembly comprising multiple inserts, e.g. comprising at least six inserts, according to one or more of the preceding claims, wherein the inserts are rigidly provided in a predetermined pattern, e.g. depending on a distribution of associated cell culture vessels, e.g. a distribution of wells in a well plate.

11. Insert assembly according to claim 10, wherein the inner spaces of the multiple inserts are interconnected, e.g. via hollow insert connectors, allowing the gas carrier to flow between the inner spaces.

12. Lid for a cell culture vessel, e.g. for a well of a well plate, comprising an insert according to one or more of the claims 5 - 9 or an insert assembly according to one or more of the claims 10 - 11.

13. Assembly for cell growth comprising:- a cell culture vessel, e.g. a well of a well plate, for containing a cell growth medium, wherein the cell culture vessel comprises a side wall surrounding the cell cultureWU R24002WO / P36805PC00 - 14 - vessel and a bottom wall displaced from a top side of the cell culture vessel by the side wall; and- an insert according to one or more of claims 5 - 9, an insert assembly according to claim 10 or 11 , or a lid according to claim 12, wherein the insert, when inserted into the cell culture vessel, at least partially extends from the top side to the bottom wall.

14. Method for growth, wherein use is made of an insert according to one or more of the claims 1 - 9, an insert assembly according to one or more of the claims 10 - 11 , a lid according to claim 12 or an assembly according to claim 13.

15. Method according to claim 14, comprising:- inserting the insert into a growth medium.

16. Method according to claim 14 or 15, comprising:- providing a flow of liquid gas-carrier in the inner space;- adding to and / or removing from the gas carrier in the inner space a predetermined amount of gas carrier; and / or- adding to and / or removing from the gas carrier in the inner space a predetermined amount of gas.

17. Method according to any of claims 14-16, wherein the method comprises removing, sterilizing and reusing the insert, e.g. wherein the insert is adapted to withstand repeated sterilization cycles.

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