WO2013043072A1 - Contenant pour une culture cellulaire en 3d dynamique - Google Patents

Contenant pour une culture cellulaire en 3d dynamique Download PDF

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Publication number
WO2013043072A1
WO2013043072A1 PCT/PT2012/000037 PT2012000037W WO2013043072A1 WO 2013043072 A1 WO2013043072 A1 WO 2013043072A1 PT 2012000037 W PT2012000037 W PT 2012000037W WO 2013043072 A1 WO2013043072 A1 WO 2013043072A1
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WO
WIPO (PCT)
Prior art keywords
container
chambers
impeller
shaft
cell samples
Prior art date
Application number
PCT/PT2012/000037
Other languages
English (en)
Inventor
Cristina Maria Santos Alves de Carvalho BARRIAS
Raquel Madeira GONÇALVES
Original Assignee
Ineb-Instituto De Engenharia Biomédica
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ineb-Instituto De Engenharia Biomédica filed Critical Ineb-Instituto De Engenharia Biomédica
Publication of WO2013043072A1 publication Critical patent/WO2013043072A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/14—Rotation or movement of the cells support, e.g. rotated hollow fibers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/18—Flow directing inserts
    • C12M27/22—Perforated plates, discs or walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures

Definitions

  • the invention relates to a container for culturing cells in a spinner flask and, in particular, relates to a multi-compartment container for three-dimensional (3D) cell culture under dynamic conditions.
  • the invention also relates to the use of the container, kits and spinner flasks comprising the container, and a method of culturing cells using the container.
  • Bioreactors can be designed in order to create spatially uniform cell distributions within 3D scaffolds, enhance the diffusion of gases, nutrients and biochemical factors from the culture medium into the construct interior, and also promote continuous removal of cellular metabolites. Moreover, within certain bioreactors, samples can additionally be exposed to appropriate physical stimuli.
  • US Patent Application Number 2010/0028992 discloses the broad concept of a spinner flask carrying a perforated cell-culturing basket.
  • this document does not disclose the use of a perforated lid to prevent free-floating samples from escaping.
  • this document does not disclose a disc-shaped holder arrangement with multiple but individual chambers for easy deposit and recovery of cell-loaded samples and establishment of 3D co-cultures. Additionally, it does not suggest the stacking of multiple containers or the modification of a standard spinner flask system, in particular, a modified impeller assembly.
  • the present invention provides a container for culturing cell samples in a 3D matrix in a spinner flask, the container comprising a plurality of chambers for containing the cell samples, wherein at least one of the walls of the chambers are perforated to allow culture media to enter the chambers through the perforations from outside the container, and wherein the container comprises one or more detachable lids which, when detached, open the top of the chambers to allow cell samples to be placed directly into or removed from the chambers.
  • the container and, in particular, the chambers in which the cell samples are cultured provides a means of protecting delicate/fragile samples from the turbulent environment generated by the stirring process, expanding the versatility of spinner- flask systems. Further, since cell samples can be loaded individually into the chambers, each sample can be manipulated over the course of culturing independently of the samples in the other chambers. This is due to the fact that the cell sample in each chamber is physically isolated from the cell samples in other chambers.
  • the container comprises a body within which a plurality of cavities are defined. These cavities form the chambers within the container once they are closed by the lid(s) of the container.
  • the cavities/chambers are distinct from one another.
  • the body of the container may be formed from a single piece of material into which the cavities (or chambers) have been formed.
  • the cavities in the body may be completely open at the top, i.e. they may not have a structure defining a surface, which closes the top of the cavity. Put another way, the face of the cavity, which would form the top, may not be present.
  • the body of the container can have the one or more detachable lids attached thereto to close the cavities, thus forming the closed chambers.
  • the one or more lids when attached to the body of the container, provide a top face to the cavities to form the closed chambers.
  • the one or more detachable lids attach to the top of the body of the container to close the top of the cavities (or chambers).
  • the chambers are opened to form open cavities into which 3D matrices of cells can be place or removed.
  • the fact that the top of each of the chambers is open allows cell samples to be loaded into the container more easily as they can be loaded without manipulating the container.
  • the presence of one or more lids allow samples to be loaded into the cavities which are then closed off with the one or more lids to form closed chambers. This is particularly advantageous as free-floating samples such as hydrogels can be placed into the chambers and cultured without the risk of the samples floating out of the chambers.
  • cavities and “chambers” have been used when referring to the portion of the container in which the cell samples are contained and cultured.
  • the term “cavity” is intended to mean that there is an open face present, for example, when the lid is detached, to allow cell samples to be inserted into or removed from the cavity.
  • the term “chamber” is intended to mean that the faces defining the chamber are present, for example, when the lid is attached, so that the cell samples in the chamber are physically isolated from the exterior of the chamber in use and cannot escape from the chamber.
  • the skilled person will appreciate that in order to put cell samples into the chamber, it is necessary to open the chamber.
  • the chamber may be referred to in its open state, for example, as an open chamber. The skilled person will realise from the context in which it is used the intended meaning of each of these terms.
  • the container is for culturing cells in a spinner flask.
  • the container should be sized so that it can fit into a spinner flask.
  • the dimensions of the container are dependent on those of the spinner flask and, in particular, the dimensions of the central opening of the spinner flask.
  • the outer diameter of the container is about 20-30 mm.
  • the outer diameter (OD) of the container is about 22-28 mm or about 24-26 mm. More preferably, the outer diameter of the container is about 25 mm.
  • the overall height (OAH) of the container may be about 4-8 mm or about 5-7 mm.
  • the overall height of the container is about 6 mm.
  • spinner flasks are widely available from a number of manufacturers. Since the container can be used in spinner flasks and, in particular, small volume spinner flasks, this allows the use of a small volume of culture media to culture the cells. This is a significant advantage as some culture media, such as culture media for stem cells, is relatively expensive.
  • the container and, in particular, the chambers in the container, are for culturing cell samples in a 3D matrix.
  • Suitable 3D matrices in which cells can be cultured can be made of polymers (natural or synthetic), ceramics, or composites.
  • the 3D matrix can be in the form of: a hydrogel, a porous 3D scaffold, a rapid-prototyping scaffold, a foam, a sponge, a mesh, microparticles, fiber-like networks, and combinations thereof, for example, microparticle-loaded hydrogels.
  • the container can also be used to culture other biological samples such as tissue biopsies, cell clusters and prototissues.
  • the cells are cultured in hydrogel-like matrices.
  • the chambers in the container can be used to culture any type of cells in a 3D matrix.
  • Such cells include attachment-dependent cells, for example, mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), endothelial cells, fibroblasts, neuronal cells, hepatocytes, condrocytes, smooth-muscle cells, cancer cells, etc.
  • Further cells include suspension cells which can be entrapped in the 3D matrix, for example, hematopoietic stem cells and different types of blood cells.
  • the chambers in the container may also be useful for the 3D culture of non-mammalian cells, such as plant, bacterial, yeast and viral cultures.
  • the container comprises a plurality of chambers for containing and culturing the cell samples.
  • the container may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chambers.
  • the container comprises six chambers.
  • Each chamber has perforations in at least one of the walls.
  • each chamber has perforations in the bottom, side and top walls of the chamber.
  • the perforations in at least one of the walls of the chambers are intended to allow culture media to enter the chambers through the perforations from outside the container. Equally, culture media can leave the chambers through the perforations returning to the outside of the container. This permits fluid perfusion through the 3D cell-material constructs which are contained in the chambers.
  • one or more of the bottom, side and top walls of the chambers can be perforated. Further, two or more of the bottom, side and top walls of the chambers can be perforated.
  • the bottom, side and top walls of the chambers have perforations therein.
  • the top wall of the chambers is formed by the one or more lids of the container. Therefore, the one or more lids can have perforations therein.
  • the perforations may be over the whole top surface of the one or more lids so that the one or more lids can be placed in any orientation while still having the perforations located in the top surface of the chambers.
  • the terms “bottom”, “side” and “top” in the context of the container are used with reference to the orientation of the container when it is in use. For example, when the lid of the container is described as attaching to the top of the container, this is the top of the container in its normal and intended orientation in use. Further, with respect to the walls of the chambers, again the terms “bottom”, “side” and “top” are used with reference to their orientation in use.
  • the size and number of perforations in the walls of the chambers should be sufficiently large to allow culture media to enter the chambers through the perforations from outside the container in a sufficient amount to allow all the cells in the 3D matrix to obtain all the nutrients they require from the culture media and to grow as desired.
  • the size of the perforations should not be so large that some or all of the cell sample in the chamber can escape through the perforations.
  • the size of the perforations should not be so large that the agitation of the culture media outside the container can exert significant shear forces on the cell samples in the chambers of the container.
  • the perforations may have a variety of shapes. The most common is the simple perforation (a simple opening), e.g. a substantially circular hole in the chamber wall.
  • the number of perforations may vary according to the chambers size. Typically, there may be about 20-30 perforations in the walls of the chamber.
  • the size (i.e. diameter or cross sectional dimensions) of the perforations may be between about 0.2 mm and about 1.2 mm. Preferably, the perforations are about 0.5- 1.0 mm. Preferably, the perforations are spherical.
  • the chambers of the container are physically isolated from each other within the container so that culture media cannot flow directly from one chamber into another chamber.
  • the perforations in the walls of the chambers only allow fluid communication between the outside of the container and the inside of the chamber. There are no perforations between the chambers. Therefore, no direct fluid communication can occur between the inside of two adjacent chambers.
  • the compartmentalisation of the samples in this way enables the establishment of more complex 3D cultures, such as indirect contact co-cultures using different types of cells sharing the same microenvironment.
  • Two different cell types can be inserted into two of the chambers. Any molecules secreted by the first cell type will be released into the culture media, i.e. the microenvironment. These molecules may then influence the culturing of the second cell type as the secreted molecules will perfuse into the chamber of the second cell type from outside the chamber.
  • the container can also be used to perform advanced 3D co-cultures, as it facilitates the biochemical communication between adjacent chambers, which may contain cells of different types maintained physically separated from one another.
  • the chambers of the container may be arranged in substantially the same horizontal plane when in use so that each of the chambers can be accessed independently when the one or more lids is detached. In other words, the chambers are positioned horizontally adjacent to each other when the container is orientated as it would be in use. As indicated above, this allows cell samples to be loaded into the container more easily as they can be loaded without manipulating the orientation of the container.
  • the chambers of the container may be any suitable size for containing 3D matrices of cells.
  • the size of the chambers depends on the overall size of the container, and the chambers can have different shapes and sizes.
  • the chambers are disc-shaped having a diameter of about 4-8 mm or about 5-7 mm.
  • the disc-shaped chamber have a diameter of about 6 mm.
  • the chambers may have a height of about 4-8 mm or about 5-7 mm.
  • the height of the chambers is about 6 mm.
  • the chambers are disc-shaped having a diameter of about 6 mm and a height of about 6 mm.
  • the container including the one or more lids and the chambers, may be made from any suitable material.
  • the container and, in particular, the chambers are made from a non-adherent material, i.e. a material with low protein binding/cell adhesion.
  • the material is preferably chemically resistant and bio-inert.
  • the container can be made of a material which can be sterilised, for example, in an autoclave, or using ethylene oxide, gamma-irradiation or ethanol. This allows repeated use of the container.
  • the container and chambers are made from a polymeric material such as polytetrafluoroethylene (PTFE).
  • the container is for use in a spinner flask. Therefore, the container may be adapted to attach to the impeller of a spinner flask. In particular embodiments, the container may be adapted to attach to the impeller assembly of a spinner flask. In some embodiments, the container is disc shaped. Further, the container may comprise a central opening which can accommodate the shaft of an impeller and hold the container on the impeller, e.g. on the shaft of the impeller. The central opening should be at the centre of gravity of the container so that when the container spins on the impeller, no vibrations are caused which could affect the culturing of the cells and/or the apparatus. Preferably, the opening is cylindrical.
  • the container comprises a central opening for positioning the container on the impeller, e.g. the shaft of the impeller, and is shaped to allow a plurality of containers to be loaded onto the impeller.
  • the containers may stack on top of each other.
  • the container is adapted so that two containers can be positioned on the impeller.
  • the invention relates to the use of the containers described above for culturing cells.
  • kit comprising:
  • a container for culturing cell samples in a 3D matrix in a spinner flask comprising a plurality of chambers for containing the cell samples, wherein at least one of the walls of the chambers are perforated to allow culture media to enter the chambers through the perforations from outside the container, and wherein the container comprises one or more detachable lids which, when detached, open the top of the chambers to allow cell samples to be placed directly into or removed from the chambers, and wherein the container comprises a central opening which can accommodate the shaft of an impeller and hold the container in position on the impeller; and
  • an impeller for a spinner flask wherein the shaft of the impeller is adapted to fit into the central opening of the container to hold the container on the impeller.
  • the container may be held on the shaft of the impeller.
  • the shaft of the impeller comprises a magnet holder.
  • the container may be held on the magnet holder.
  • the central opening of the container can accommodate the shaft and/or magnet holder of an impeller and hold the container in position on the impeller.
  • the magnet holder and/or shaft of the impeller may accommodate a plurality of containers, for example, two container.
  • the central opening of the container may be cylindrical.
  • the shaft of the impeller may have a first portion which has a diameter of less than the diameter of the central opening of the container and a second portion which has a diameter of equal to or more than the diameter of the central opening of the container, wherein the container fits onto the first portion of the shaft and is held on the shaft by the second portion of the shaft as the container cannot move onto the second portion of the shaft due to diameter of the second portion of the shaft being the same as or more than the central opening of the container.
  • the magnet holder of the impeller may have a first portion which has a diameter of less than the diameter of the central opening of the container and a second portion which has a diameter of equal to or more than the diameter of the central opening of the container, wherein the container fits onto the first portion of the magnet holder and is held on the magnet holder by the second portion of the magnet holder as the container cannot move onto the second portion of the magnet holder due to diameter of the second portion of the magnet holder being the same as or more than the central opening of the container.
  • a spinner flask assembly for culturing cell samples in a 3D matrix, the assembly comprising:
  • a container for culturing cell samples in a 3D matrix comprising a plurality of chambers for containing the cell samples, wherein at least one of the walls of the chambers are perforated to allow culture media to enter the chambers through the perforations from outside the container, and wherein the container comprises one or more detachable lids which, when detached, open the top of the chambers to allow cell samples to be placed directly into or removed from the chambers, and wherein the container comprises a central opening which can accommodate the shaft of an impeller and hold the container in position on the impeller;
  • the impeller shaft may comprise a magnet holder adapted to fit into the central opening of the container to hold the container on the impeller.
  • the invention provides a method of culturing cells comprising: inserting a cell sample in a 3D matrix into a chamber of a container, wherein the container comprises a plurality of chambers for containing cell samples, wherein at least one of the walls of the chambers are perforated to allow culture media to enter the chambers through the perforations from outside the container, and wherein the container comprises one or more detachable lids which, when detached, open the top of the chambers to allow cell samples to be placed directly into or removed from the chambers; and
  • the method comprises inserting a cell sample into more than one of the plurality of chambers of the container. Further, the method may comprise inserting a cell sample into each of the plurality of chambers of the container. In further embodiments, the method may comprise inserting a sample of a first type of cells into a first chamber of the container and inserting a sample of a second type of cells into a second chamber of the container. This allows the co-culture of two different cell types and allows the two different cell types to be tested under the same conditions. Optionally, further different cell types can be place in the other chambers to allow co-culture of more than two different cell types.
  • Figure 1 is a schematic representation (top and side views) of one multi-compartment container without the lid. One side of each of the cavities in the container and the bottom wall of the cavities are perforated.
  • Figure 2 is a schematic representation (top and side views) of the lid for a container. The top of the lid is perforated.
  • Figure 3 is a schematic representation (top, side, front and bottom views) of a magnet holder which can form part of the impeller shaft.
  • Figure 4 is a schematic representation (side view) of an impeller shaft with a magnet holder.holding two containers and a lid.
  • the magnet holder of the impeller of the spinner flask has been modified to accommodate the two containers.
  • Figure 5 shows two multi-compartment containers with a lid assembled in a spinner flask.
  • Figure 6 shows representative hematoxylin/eosin (H&E)-stained cryo-sections of cell- loaded 3D scaffolds cultured in standard static conditions (A 1-3) and in the multicompartment holder under dynamic conditions in a spinner flask (B 1-3). Cell distribution within 3D scaffolds in both conditions was analysed at the construct periphery (Al , Bl) and core (A2, B2, A3, B3).
  • the container Fig.
  • a 3D matrix for culturing cells in a 3D matrix comprises a multicompartment chamber, adaptable to small volume spinner-flasks such as 25 raL spinner flasks with dimensions of: ODxOAH: 38x 120 mm).
  • the unit is machined from the polymer PTFE.
  • Each container (ODxOAH: 25 6mm) has six independent compartments (6 mm diameter and 6 mm height) to house disc-shaped samples with a maximum size of 5 mm diameter and 5 mm height.
  • the compartments are perforated (top, bottom and side, perforation size 1 mm) to permit fluid perfusion through the 3D cell-material constructs.
  • the container has a perforated lid (Fig. 2)) to retain free- floating samples.
  • the PTFE magnet holder (Fig. 3) of the spinner- flask impeller assembly (Fig. 4) was redesigned to better accommodate the sample-holding containers, specifically the diameter of the first portion of the shaft was reduced to 1 1 mm.
  • the total volume of culture media needed to submerge the whole assembly is less than 25mL.
  • the whole assembly (Fig. 5) can be sterilized in an autoclave.
  • the impeller assembly (Fig. 4) is composed of a shaft (1) with a magnet holder (2), and a magnet (3).
  • the magnet holder has a first portion which has a diameter of 1 1 mm, less than the diameter of the central opening of the containers, and a second portion which has a diameter of 16 mm, more than the diameter of the central opening of the containers.
  • the containers (4) fit onto the first portion of the magnet holder and are held on the magnet holder by the second portion of the magnet holder.
  • a lid (5) can be assembled on the top of the containers.
  • a magnet is inserted in the magnet holder.
  • the impeller assembly with the containers and lid can be mounted in a spinner flask (6) (Fig. 5). The assembly is placed on a magnetic stirrer inside an incubator.
  • hMSCs human mesenchymal stem cells
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • P/S Penicillin/Streptomycin
  • hMSCs were seeded in Chitosan 3D cylindrical porous scaffolds with 4 mm diameter and 3 mm height, and a pore diameter in the range 100-300 ⁇ ⁇ .
  • hMSCs-loaded scaffolds were fixed in 4% paraformaldehyde and embedded in cryomatrix freezing medium. Scaffolds were then sectioned (30 ⁇ ) into slides. Sections were stained with H&E and images were collected at the core and periphery of the scaffold .
  • Al to A3 correspond to cell-loaded 3D scaffolds cultured in standard static conditions, while Bl to B3 refers to cell cultures in the multi-compartment holder under dynamic conditions in a spinner flask.
  • the invention relates to a multi-compartment container for culturing cells in 3D matrices under dynamic conditions in a spinner flask.
  • the main advantages of the system are to:
  • the compartments in which the cell samples are cultured provide a means of protecting fragile samples from the turbulent environment generated by the stirring process, expanding the versatility of spinner-flask systems. Moreover, the multi- compartment container allows for a same-time, same-media culture of multiple samples. These represent enormous advantages over traditional spinner flask systems, along with the following product features:
  • the product can be easily adapted to fit different types of spinner flasks design (from different manufacturers);
  • the product presents multiple compartments, allowing for a large number of experimental conditions to be tested simultaneously (e.g. testing different types of 3D scaffolds, materials functionalization, etc.); - It allows for each compartment to be treated separately, permitting that a sample can be removed at specific times during the course of an experiment;
  • the container leads to sizeable saving on media, size of scaffold necessary and time.

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Abstract

L'invention concerne un contenant qui permet la culture d'échantillons cellulaires dans une matrice en 3D dans un flacon d'agitation, le contenant comportant une pluralité de chambres pour contenir les échantillons cellulaires, au moins une des parois des chambres étant perforée afin de permettre aux milieux de culture d'entrer dans les chambres par l'intermédiaire des perforations, à partir de l'extérieur du contenant, le contenant comportant un ou plusieurs couvercles détachables qui, lorsqu'ils sont détachés, ouvrent le sommet des chambres pour permettre aux échantillons cellulaires d'être placés directement dans les chambres ou retirés de celles-ci.
PCT/PT2012/000037 2011-09-22 2012-09-21 Contenant pour une culture cellulaire en 3d dynamique WO2013043072A1 (fr)

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GB201116397A GB201116397D0 (en) 2011-09-22 2011-09-22 Container
GB1116397.9 2011-09-22

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WO2016089852A1 (fr) * 2014-12-02 2016-06-09 Histogenics Corporation Récipient pour culture cellulaire et tissulaire
US10077420B2 (en) 2014-12-02 2018-09-18 Histogenics Corporation Cell and tissue culture container
US11555172B2 (en) 2014-12-02 2023-01-17 Ocugen, Inc. Cell and tissue culture container

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