Patent Application
20240287427
The technical field generally relates to cell culture techniques. More particularly, the technical field relates to cell culture techniques for preparing compartmentalized in vitro models with a biological material that includes an elongated component.
Models that can predict human or animal responses to various chemical and biological compounds, such as medications, pesticides, and cosmetics, and that are easily scalable to test multiple compounds already on the market and new products launched each year are desirable.
In vitro models can be used to refine, reduce, and replace animal testing, for instance in the pharmaceutical, chemical and cosmetic industries. In vitro models can offer opportunities for performing various experiments on biological materials. These experiments can be performed for instance in relation to toxicity testing, efficacy testing, drug screening, drug repurposing, disease modelling, etc.
However, conventional techniques for modeling biological tissues in vitro do not enable compartmentalization of components of a biological tissue or do not enable obtaining an organized architecture of the components of the biological tissue. For instance, hair or neurons are typically grown according to an unorganized network, which can inaccurately model such biological materials and render the model unsuitable for performing certain tests on specific components of the biological material.
Accordingly, there remain a number of challenges with respect to the production of in vitro models of biological materials.
In accordance with an aspect, there is provided a cell culture device for preparing a compartmentalized in vitro model using a biological material having a multicellular component and an elongated component, the cell culture device comprising:
In some implementations, the channel comprises a converging portion that converges inwardly toward the outlet reservoir.
In some implementations, the converging portion defines at least a portion of the multicellular component receiving portion of the channel.
In some implementations, the converging portion defines at least a portion of the elongated component receiving portion of the channel.
In some implementations, the converging portion includes an inwardly converging top wall that converges inwardly toward a center of the channel.
In some implementations, the inwardly converging top wall defines a step change.
In some implementations, the inwardly converging top wall includes a curvature.
In some implementations, the channel has a width that is substantially constant.
In some implementations, the converging portion includes an inwardly converging sidewall that converges inwardly toward a center of the channel.
In some implementations, the inwardly converging sidewall defines a step change.
In some implementations, the inwardly converging sidewall includes a curvature.
In some implementations, the converging portion comprises a frustoconical converging portion.
In some implementations, the converging portion comprises a frustopyramidal converging portion.
In some implementations, the converging portion comprises a neck portion to contribute to stabilizing the elongated component of the biological material.
In some implementations, the converging portion comprises an inwardly protruding member to contribute to stabilizing the elongated component of the biological material.
In some implementations, the channel further comprises a tubular portion provided downstream of the converging portion, the tubular portion having a substantially constant diameter throughout its length.
In some implementations, the converging portion of the channel comprises converging sections each converging at a corresponding angle toward the outlet reservoir.
In some implementations, the converging sections successively comprises a first converging section and a second converging section, the corresponding angle of the first converging section being larger than the corresponding angle of the second converging section.
In some implementations, a transition between successive ones of the converging sections comprises a curved transition.
In some implementations, a transition between successive ones of the converging sections comprises a sharp transition.
In some implementations, the channel is sized and configured to maintain the elongated component of the biological material in a substantially elongated configuration.
In some implementations, the channel comprises a plurality of channels.
In some implementations, adjacent channels of the plurality of channels are similar to each other.
In some implementations, at least one channel of the plurality of channels is configured differently than at least one other channel of the plurality of channels.
In some implementations, the inlet reservoir comprises a plurality of inlet reservoirs.
In some implementations, at least one channel of the plurality of channels is in fluid communication with a corresponding inlet reservoir of the plurality of inlet reservoirs.
In some implementations, the outlet reservoir comprises a plurality of outlet reservoirs.
In some implementations, at least one channel of the plurality of channels is in fluid communication with a corresponding outlet reservoir of the plurality of outlet reservoirs.
In some implementations, the cell culture layer is configured to extend substantially horizontally, the inlet reservoir and the outlet reservoir being provided in a longitudinally spaced-apart relationship.
In some implementations, the cell culture layer further comprises an inlet well in fluid communication with the inlet reservoir, the inlet well being configured to receive the inlet fluid medium therein.
In some implementations, the cell culture layer comprises an inlet manifold extending between the inlet well and the inlet reservoir, the inlet manifold comprising a plurality of inlet reservoir channels for directing flow of the inlet fluid medium from the inlet well to the inlet reservoir with reduced turbulence.
In some implementations, the cell culture layer comprises an inlet reservoir channel extending between the inlet well and the inlet reservoir for directing flow of the inlet fluid medium into the inlet reservoir with reduced turbulence.
In some implementations, the inlet reservoir channel is a converging inlet reservoir channel converging toward the inlet reservoir.
In some implementations, the inlet reservoir channel comprises at least one turbulence reducing feature.
In some implementations, the cell culture layer further comprises an outlet well in fluid communication with the outlet reservoir, the outlet well being configured to receive the outlet fluid medium therein.
In some implementations, the cell culture layer comprises an outlet manifold extending between the outlet reservoir and the outlet well, the outlet manifold comprising a plurality of outlet reservoir channels for directing flow of the outlet fluid medium from the outlet reservoir to the outlet well with reduced turbulence.
In some implementations, the cell culture layer comprises an outlet reservoir channel extending between the outlet reservoir and the outlet well for directing flow of the outlet fluid medium from the outlet reservoir to the outlet well with reduced turbulence.
In some implementations, the outlet reservoir channel is a converging outlet reservoir channel converging toward the outlet well.
In some implementations, the outlet reservoir channel comprises at least one turbulence reducing feature.
In some implementations, the cell culture layer is configured to extend substantially horizontally, the inlet reservoir and the outlet reservoir being provided in a superposed relationship, and the converging portion being a downwardly converging portion.
In some implementations, the outlet reservoir is U-shaped.
In some implementations, the inlet reservoir comprises a plurality of inlet reservoirs, and adjacent ones of the plurality of inlet reservoirs are in fluid communication via an inlet reservoir bridge extending therebetween.
In some implementations, the cell culture device further comprises a cover configured to be positionable on an upper surface of the cell culture layer.
In some implementations, the cover is configured to provide a fluid tight closure for the inlet reservoir once positioned on the upper surface of the cell culture layer.
In some implementations, the cover comprises a microporous membrane.
In some implementations, the cover comprises a collagen membrane.
In some implementations, the cell culture device further comprises a biological model positionable on an upper surface of the cell culture layer.
In some implementations, the biological model is positionable on the cell culture layer to enable interaction with the biological material received in the channel.
In some implementations, the biological model comprises cultured cells.
In some implementations, the biological model comprises a biological tissue.
In some implementations, the biological model comprises a biological tissue model.
In some implementations, the biological tissue model comprises a three-dimensional skin model.
In some implementations, the cell culture device further comprises an inlet well feeding system in fluid communication with the inlet well to supply additional fluid culture medium to the inlet well.
In some implementations, the cell culture plate is a petri dish.
In some implementations, the cell culture device complies with American National Standards Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) microplate standards.
In some implementations, the multicellular component of the biological material comprises a cluster of neuronal cell bodies and the elongated component comprising axons.
In some implementations, the multicellular component of the biological material comprises a follicle and the elongated component comprising a hair.
In accordance with another aspect, there is provided a cell culture device for preparing a compartmentalized in vitro model using a biological material having a multicellular component and an elongated component, the cell culture device comprising:
In some implementations, the electrode forms part of an electrode layer.
In some implementations, the electrode layer is positionable underneath the cell culture layer.
In some implementations, the electrode layer is receivable onto an upper surface of the cell culture layer.
In some implementations, the cell culture device further comprises a biological model receivable on an upper surface of the cell culture layer.
In some implementations, the electrode layer is provided onto the biological model.
In some implementations, the electrode layer is provided as part of the biological model.
In some implementations, the electrode comprises a plurality of electrodes.
In some implementations, the channel comprises a plurality of channels.
In some implementations, the plurality of electrodes are distributed over the electrode layer in accordance with a configuration of the plurality of channels.
In some implementations, the electrode is located in an adjacent reservoir.
In some implementations, the electrode comprises at least one of a metallic electrode, a metal oxide electrode, a carbon electrode, a multi-electrode array, and a field effect transistor detector.
In some implementations, the electrode is configured for stimulating the biological material.
In some implementations, the electrode is configured for at least one of collecting, recording, measuring, or detecting a response of the biological material to stimulation.
In some implementations, the cell culture device further comprises an electronic device in ohmic connection with the electrode.
In some implementations, the electronic device comprises a sensing device.
In some implementations, the electronic device comprises a stimulating device.
In some implementations, the electronic device is configured for providing an electrical read-out comprising at least one of a potential recording, an impedance spectroscopy recording, a voltammetry recording and an amperometry recording.
In some implementations, the cell culture device further comprises a sensor configured for stimulating the biological material, measuring a response from the biological material to stimulation, providing an outlet or receiving an inlet.
In some implementations, the sensor comprises an optical or an electrical transducer.
In accordance with another aspect, there is provided a method for preparing a compartmentalized in vitro model of a biological material that includes a multicellular component and an elongated component using a cell culture layer receivable in a cell culture plate, the method comprising:
In some implementations, the channel comprises a converging portion that converges inwardly.
In some implementations, the cell culture layer further comprises an outlet reservoir in fluid communication with the channel, with the channel extending between the inlet reservoir and the outlet reservoir.
In some implementations, at least a portion of the elongated component of the biological material extend within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on the at least a portion of the elongated component of the biological material.
In some implementations, the method further comprises positioning an additional biological material within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on additional biological material.
In some implementations, the method further comprises adding an inlet test substance to the inlet reservoir to perform testing on the non-elongated component of the biological material.
In some implementations, the method further comprises placing a cover on an upper surface of the cell culture layer.
In some implementations, the cover comprises a microporous membrane.
In some implementations, the cover comprises a collagen membrane.
In some implementations, the method further comprises placing a biological model on an upper surface of the cell culture layer.
In some implementations, the biological model is positionable on the cell culture layer to enable interaction with the biological material received in the cell culture layer.
In some implementations, the multicellular component of the biological material comprises follicles.
In some implementations, the elongated component of the biological material comprises hair.
In some implementations, the multicellular component of the biological material comprises cell bodies of neurons.
In some implementations, the biological material is provided as a neurosphere.
In some implementations, the elongated component of the biological material comprises axons.
In accordance with another aspect, there is provided a method for preparing a compartmentalized in vitro model of a biological material using a cell culture layer receivable in a cell culture plate, the method comprising:
In some implementations, the channel comprises a converging portion that converges inwardly toward the outlet reservoir.
In some implementations, the cell culture layer further comprises an outlet reservoir in fluid communication with the channel, with the channel extending between the inlet reservoir and the outlet reservoir.
In some implementations, at least a portion of the second biological material extends within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on the second biological material.
In some implementations, the method further comprises positioning an additional biological material within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on the additional biological material.
In some implementations, the method further comprises adding an inlet test substance to the inlet reservoir to perform testing on the first biological material.
In some implementations, the method further comprises placing a cover on an upper surface of the cell culture layer.
In some implementations, the cover comprises a microporous membrane.
In some implementations, the cover comprises a collagen membrane.
In some implementations, the method further comprises placing a biological model on an upper surface of the cell culture layer.
In some implementations, the biological model is positionable on the cell culture layer to enable interaction with the biological material received in the cell culture layer.
In some implementations, the first biological material comprises neurons.
In some implementations, the neurons are provided as neurospheres.
In some implementations, the elongated component of the second biological material comprises axons.
In some implementations, the first biological material comprises follicles.
In some implementations, the second biological material comprises hairs.
In some implementations, the additional biological material comprises at least one of heart tissue, intestinal tissue, muscle tissue, and corneal tissue.
In accordance with another aspect, there is provided a cell culture device for preparing a compartmentalized in vitro model using a biological material having a multicellular component and an elongated component, the cell culture device comprising:
In some implementations, the channel comprises a converging portion that converges inwardly toward the outlet reservoir.
In some implementations, the converging portion includes an inwardly converging top wall that converges inwardly toward a center of the channel.
In some implementations, the inwardly converging top wall defines a step change.
In some implementations, the inwardly converging top wall includes a curvature.
In some implementations, the channel has a width that is substantially constant.
In some implementations, the converging portion includes an inwardly converging sidewall that converges inwardly toward a center of the channel.
In some implementations, the inwardly converging sidewall defines a step change.
In some implementations, the inwardly converging sidewall includes a curvature.
In some implementations, the converging portion comprises a frustoconical converging portion.
In some implementations, the converging portion comprises a frustopyramidal converging portion.
In some implementations, the converging portion comprises a neck portion to contribute to stabilizing the elongated component of the biological material.
In some implementations, the converging portion comprises an inwardly protruding member to contribute to stabilizing the elongated component of the biological material.
In some implementations, the channel further comprises a tubular portion provided downstream of the converging portion, the tubular portion having a substantially constant diameter throughout its length.
In some implementations, the converging portion of the channel comprises converging sections each converging at a corresponding angle toward the outlet reservoir.
In some implementations, the converging sections successively comprises a first converging section and a second converging section, the corresponding angle of the first converging section being larger than the corresponding angle of the second converging section.
In some implementations, a transition between successive ones of the converging sections comprises a curved transition.
In some implementations, a transition between successive ones of the converging sections comprises a sharp transition.
In some implementations, the channel is sized and configured to maintain the elongated component of the biological material in a substantially elongated configuration.
In some implementations, the channel comprises a plurality of channels.
In some implementations, adjacent channels of the plurality of channels are similar to each other.
In some implementations, at least one channel of the plurality of channels is configured differently than at least one other channel of the plurality of channels.
In some implementations, the inlet reservoir comprises a plurality of inlet reservoirs.
In some implementations, at least one channel of the plurality of channels is in fluid communication with a corresponding inlet reservoir of the plurality of inlet reservoirs.
In some implementations, the outlet reservoir comprises a plurality of outlet reservoirs.
In some implementations, at least one channel of the plurality of channels is in fluid communication with a corresponding outlet reservoir of the plurality of outlet reservoirs.
In some implementations, the cell culture layer is configured to extend substantially horizontally, the inlet reservoir and the outlet reservoir being provided in a longitudinally spaced-apart relationship.
In some implementations, the cell culture layer further comprises an inlet well in fluid communication with the inlet reservoir, the inlet well being configured to receive the inlet fluid medium therein.
In some implementations, the cell culture layer comprises an inlet manifold extending between the inlet well and the inlet reservoir, the inlet manifold comprising a plurality of inlet reservoir channels for directing flow of the inlet fluid medium from the inlet well to the inlet reservoir with reduced turbulence.
In some implementations, the cell culture layer comprises an inlet reservoir channel extending between the inlet well and the inlet reservoir for directing flow of the inlet fluid medium into the inlet reservoir with reduced turbulence.
In some implementations, the inlet reservoir channel is a converging inlet reservoir channel converging toward the inlet reservoir.
In some implementations, the inlet reservoir channel comprises at least one turbulence reducing feature.
In some implementations, the cell culture layer further comprises an outlet well in fluid communication with the outlet reservoir, the outlet well being configured to receive the outlet fluid medium therein.
In some implementations, the cell culture layer comprises an outlet manifold extending between the outlet reservoir and the outlet well, the outlet manifold comprising a plurality of outlet reservoir channels for directing flow of the outlet fluid medium from the outlet reservoir to the outlet well with reduced turbulence.
In some implementations, the cell culture layer comprises an outlet reservoir channel extending between the outlet reservoir and the outlet well for directing flow of the outlet fluid medium from the outlet reservoir to the outlet well with reduced turbulence.
In some implementations, the outlet reservoir channel is a converging outlet reservoir channel converging toward the outlet well.
In some implementations, the outlet reservoir channel comprises at least one turbulence reducing feature.
In some implementations, the cell culture layer is configured to extend substantially horizontally, the inlet reservoir and the outlet reservoir being provided in a superposed relationship, and the converging portion being a downwardly converging portion.
In some implementations, the outlet reservoir is U-shaped.
In some implementations, the inlet reservoir comprises a plurality of inlet reservoirs, and adjacent ones of the plurality of inlet reservoirs are in fluid communication via an inlet reservoir bridge extending therebetween.
In some implementations, the cell culture device further comprises a cover configured to be positionable on an upper surface of the cell culture layer.
In some implementations, the cover is configured to provide a fluid tight closure for the inlet reservoir once positioned on the upper surface of the cell culture layer.
In some implementations, the cover comprises a microporous membrane.
In some implementations, the cover comprises a collagen membrane.
In some implementations, the cell culture device further comprises a biological model positionable on an upper surface of the cell culture layer.
In some implementations, the biological model is positionable on the cell culture layer to enable interaction with the biological material received in the channel.
In some implementations, the biological model comprises cultured cells.
In some implementations, the biological model comprises a biological tissue.
In some implementations, the biological model comprises a biological tissue model.
In some implementations, the biological tissue model comprises a three-dimensional skin model.
In some implementations, the cell culture device further comprises an inlet well feeding system in fluid communication with the inlet well to supply additional fluid culture medium to the inlet well.
In some implementations, the cell culture plate is a petri dish.
In some implementations, the cell culture device complies with American National Standards Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) microplate standards.
In some implementations, the multicellular component of the biological material comprises a cluster of neuronal cell bodies and the elongated component comprising axons.
In some implementations, the multicellular component of the biological material comprises a follicle and the elongated component comprising a hair.
In accordance with another aspect, there is provided a method for preparing a compartmentalized in vitro model of a biological material that includes a multicellular component and an elongated component using a cell culture layer receivable in a cell culture plate, the method comprising:
In some implementations, the channel comprises a converging portion that converges inwardly.
In some implementations, the cell culture layer further comprises an outlet reservoir in fluid communication with the channel, with the channel extending between the inlet reservoir and the outlet reservoir.
In some implementations, at least a portion of the elongated component of the biological material extend within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on the at least a portion of the elongated component of the biological material.
In some implementations, the method further comprises positioning an additional biological material within the outlet reservoir.
In some implementations, the method further comprises adding an outlet test substance to the outlet reservoir to perform testing on additional biological material.
In some implementations, the method further comprises adding an inlet test substance to the inlet reservoir to perform testing on the non-elongated component of the biological material.
In some implementations, the method further comprises placing a cover on an upper surface of the cell culture layer.
In some implementations, the cover comprises a microporous membrane.
In some implementations, the cover comprises a collagen membrane.
In some implementations, the method further comprises placing a biological model on an upper surface of the cell culture layer.
In some implementations, the biological model is positionable on the cell culture layer to enable interaction with the biological material received in the cell culture layer.
In some implementations, the multicellular component of the biological material comprises follicles.
In some implementations, the elongated component of the biological material comprises hair.
In some implementations, the multicellular component of the biological material comprises cell bodies of neurons.
In some implementations, the biological material is provided as a neurosphere.
In some implementations, the elongated component of the biological material comprises axons.
In some implementations, the method further comprises one or more features as defined herein and/or as described and/or illustrated herein.
The attached figures illustrate various features, aspects and implementations of the technology described herein.
Techniques described herein relate to the development of compartmentalized in vitro models, i.e., in vitro models of a biological material that includes a multicellular component and an elongated component, the elongated component being cultured according to a given architecture such that the elongated component can maintain its elongated configuration during growth and subsequent testing, as well as extend and grow extensions inside one or more environments. An example of such compartmentalized in vitro model is a hair that extends from a hair follicle grown in a liquid environment that is compatible with culture of cells present in the hair follicle, with the hair being exposed to an air environment. Maintaining an elongated configuration of the elongated component of the biological material can contribute to more accurately mimic in vivo behavior of the biological material, and can also enable performing distinct testing on a given component of the biological material while maintaining a biological interaction between the respective components of the biological material. In addition, compartmentalized in vitro models as described herein can enable two or more types of biological material to be placed in sufficiently close proximity to enable the biological interaction therebetween to be modelled.
As used herein, the expression “multicellular component” can refer to multicellular structures such as tissues, organs, organisms, organoids, spheroids, neurospheroids, and any other biological structure or clusters or cells where two or more cells, of same cell type or different cell types, are assembled.
A biological material that can be modeled using compartmentalized in vitro models can include a particular cell type, such as neurons, or can include multiple cell types, for examples neurons and glia. It can also include specific structures such as follicles and hairs. In these examples, the axons of the neurons and the hairs can each be considered as an elongated component of a biological material. Such biological material can also be combined with other biological materials to further mimic certain complex biological tissues and produce for instance innervated skin, skin with hairs, innervated intestines, innervated heart, innervated muscle, innervated cornea, etc. Thus, the compartmentalized in vitro models can be obtained for a biological material that includes an elongated material and at least another structure that are each being compartmentalized, such as neurons that include cell bodies, and axons as an elongated component, or a hair that includes a follicle and the hair itself as the elongated component, or between two biological materials with at least one of them including an elongated component, such as between heart tissue and neurons. Cells of various other organs can also be modelled.
The compartmentalized in vitro models as described herein can be used for a wide range of cellular assays including compound screening, compound discovery, safety, efficacy testing, etc. The organized architecture of a biological material or biological materials that can be obtained by implementing the compartmentalized in vitro model as described herein can offer multiple opportunities for performing various tests on a biological material cultured according to an in vitro model that more closely resemble the characteristics of a given animal or human biological tissue compared to conventional non-compartmentalized in vitro models.
The techniques described herein in relation to the preparation of compartmentalized in vitro models involve a cell culture device that includes a cell culture layer that can be inserted into a receptacle of a cell culture plate such as those that are widely available on the market. The cell culture layer can take various forms, and generally includes designated compartments each configured for receiving a given component of a biological material or a given biological material.
For instance, the compartmentalized in vitro model can include distinct reservoirs that are in fluid communication with each other via at least one channel, the channel being configured for orienting an elongated component of the biological material or of one of the biological materials. In some implementations, the channels can also be configured to contribute to maintaining the elongated component in an elongated configuration.
The reservoirs can enable contact of a given component of the biological material with a test substance, which can be the same or be different depending on the reservoir. A response of the given component of the biological material to the test substance can then be analyzed. The presence of distinct reservoirs can facilitate the analysis of a response from each component of the biological material to a test substance, and the analysis of the interaction of the components of the biological material to one or more test substances.
Furthermore, when the compartmentalized in vitro model includes two types of biological materials, such as neurons and skin, or neurons and muscles, for instance, each of the biological materials can be tested with a test substance, which can be the same or be different depending on the reservoir, and a response of each of the biological materials to the one or more test substances can be analyzed. The interaction between the biological materials in response to the one or more test substances can also be analyzed.
The use of such compartmentalized in vitro models can enable the generation of predictive data of compounds' safety and efficacy prior to exposure to humans, and can enable the pharmaceutical, chemical, and cosmetic industries to perform reproducible and faster drug screening, toxicity, and efficacy testing and in a more cost-effectively approach compared to conventional technologies. The miniaturization of tests performed using a compartmentalized in vitro model as described herein can also enable reducing the use of reagents per experiment. Furthermore, high throughput (HT) testing of multiple compounds using a compartmentalized in vitro model as described herein can facilitate clinical translatability, thereby predicting the efficacy and toxicity of compounds faster and more efficiently.
It will be appreciated that positional descriptions such as “above”, “below”, “left”, “right”, “inwardly”, “outwardly”, “vertical” and the like should, unless otherwise indicated, be taken in the context of the figures, and should not be considered limiting. When referring to a length, for instance in the context of a length of an axon, it is to be understood that it refers to a measure along a horizontal axis. When referring to a height, for instance in the context of a height of a channel of a microfluidic layer as described herein, it is to be understood that it refers to a measure along a vertical axis. The term “outwardly” is intended to refer to a feature that extends towards an exterior side of a reference axis. The term “inwardly” is intended to refer to a feature that extends towards an interior side of a reference axis.
Various implementations of the cell culture device will now be described in greater detail.
With reference to
The cell culture device 20 can be configured to be inserted into a receptacle of a cell culture plate 32, e.g., into a cell culture dish, such as shown in
Each of the components of the cell culture layer 22 will now be described in further detail in the paragraphs below.
The cell culture layer 22 has a thickness defined between a top surface and a bottom surface thereof. In some implementations, the cell culture layer 22 can have a thickness ranging for instance from about 0.05 mm to about 100 mm. The cell culture layer 22 can be configured to be placed onto a cell culture layer-receiving surface of a cell culture plate 32. The cell culture layer 22 can be reversibly or irreversibly attached to the cell culture layer-receiving surface using any suitable method or technique, including but not limited to, compression, surface adhesion, ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding or adhesive bonding using glue or double adhesive tape.
Alternatively, and as mentioned above, the cell culture layer 22 can be integral to, e.g., moulded with, the cell culture plate 32.
The cell culture layer 22 can have various sizes and configurations. The size and configuration of the cell culture layer 22 can be adapted to the size and configuration of the receptacle or well of the cell culture plate 32 into which the cell culture layer 22 is intended to be inserted. For instance, in implementations where the cell culture layer 22 is intended to be inserted into a petri dish, the size of the cell culture layer 22 can be determined to fit within the receptacle formed by the petri dish, as shown in
The size and configuration of the cell culture layer 22 can also depend on its intended use, which can influence for instance the number of inlet reservoirs 24, the number of outlet reservoirs 26, and/or the number of channels 28, as well as the spatial distribution and size of each of these features of the cell culture layer 22.
The cell culture layer 22 can be made of any suitable polymeric material into which it is possible to carve, stamp or mold the one or more inlet reservoirs 24, the one or more outlet reservoirs 26, and the one or more channels 28. Examples of materials that can be suitable to produce the cell culture layer 22 can include, but are not limited to, polystyrene (PS), cyclo-olefin-copolymer (COC), cycloolefin polymer (COP), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene (PP), polyether ether ketone (PEEK), Teflon®, polydimethylsiloxane (PDMS), and/or thermoplastic elastomer (TPE), as well as synthetic and biological materials such hydrogels, gelatin, collagen, chitosan, etc. In some implementations, the cell culture layer 22 can be made of a polymeric material that is transparent to light in order to facilitate optical analysis and visualization of the biological material and associated elongated component extending within the channels 28.
In some implementations, the cell culture layer 22 can be fabricated integral with the bottom wall 34 of the cell culture plate 32. Examples of materials that can be suitable to fabricate the cell culture layer 22 integral with the bottom wall 34 of the cell culture plate 32 can include, but are not limited to, polystyrene (PS), cyclo-olefin-copolymer (COC), cycloolefin polymer (COP), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene or polyether ether ketone (PEEK), Teflon®, polydimethylsiloxane (PDMS), and/or thermoplastic elastomer (TPE), as well as synthetic and biological materials such hydrogels, gelatin, collagen, chitosan, etc.
The cell culture layer 22 includes at least one inlet reservoir 24 and can include at least one outlet reservoir 26. In the implementation shown in
The inlet reservoir 24 is configured to receive an inlet fluid therein. In some implementations, the inlet reservoir 24 can be further configured to receive a biological material, or a component of a biological material, therein. The inlet fluid can be a cell culture medium that enables survival and/or proliferation of the biological material or the component of the biological material received in the compartments of the cell culture layer 22, such as in the inlet reservoir 24 and/or in the channel 28. In some implementations, the inlet reservoir 24 is further configured to receive a test substance therein, such that the biological material or the component of the biological material present in the inlet reservoir and/or in the channel can be exposed to such test substance. As used herein, a test substance can be any type of substance that is desired to be tested to evaluate a response of a component of the biological material to that test substance. The test substance can take many forms, and can be for instance a liquid, a cream, a paste, an oil, a suspension, etc.
The outlet reservoir 26 can be configured to receive an outlet fluid therein. The outlet fluid can be the same as the inlet fluid or can be different from the inlet fluid. The outlet fluid can be a cell culture medium that enables survival and/or proliferation of the biological material or the component of the biological material received in the compartments of the cell culture layer 22, such as in the outlet reservoir 26 and/or in the channel 28. Alternatively, the outlet reservoir 26 can be configured to provide a substantially “dry” environment, such that the component of the biological material that eventually reaches the outlet reservoir 26 can be exposed to air. An outlet reservoir that enables exposing a component of the biological material to air can be suitable for instance when the biological material is hair. In such implementations, the hair follicle can be added to the inlet reservoir 24 or into a portion of channel 28 into which a cell culture medium is present, and the hair shaft can be inserted into the channel 28. The hair can then grow and can subsequently reach the outlet reservoir 26 with a portion of the hair being exposed to air.
In the implementations illustrated in
In some implementations, the outlet reservoir 26 is further configured to receive a test substance therein, such that the biological material present in the inlet reservoir is exposed to such test substance. As mentioned above, a test substance can be any type of substance that is desired to be tested to evaluate a response of the biological material to that test substance. The test substance can take many forms, and can be for instance a liquid, a cream, a paste, an oil, a suspension, etc.
The cell culture layer 22 can thus enable performing testing of substances in selected compartments of the cell culture layer 22 such as the inlet reservoir 24 and the outlet reservoir 26. The test substances can have different viscosities and/or solubilities depending on the compartment into which it is added. For instance, a cream can be added to one of the inlet reservoirs or the outlet reservoir, and a cell medium can be added to the remaining reservoir. Another example is an oil compound that can be added to one of the inlet reservoirs or the outlet reservoir, and a water-based compound that can be added to the remaining reservoir.
In some implementations, either one of the inlet reservoir 24 and the outlet reservoir 26 can be a closed reservoir, e.g., a reservoir that includes a top wall.
As the inlet reservoir 24 and the outlet reservoir 26 are in fluid communication with each other via the one or more channels 28, the inlet fluid and the outlet fluid can end up contacting each other at a certain location along a length of the cell culture layer 22, the length of the cell culture layer 22 being considered as extending along the y axis in the Figures. The meeting point of the inlet fluid and the outlet fluid can vary along the length of the cell culture layer 22, for instance depending on the configuration of the channels 28, and depending on whether the cell culture layer 22 is configured as a sloping cell culture layer. In particular, although referring to the cell culture layer 22 as extending substantially horizontally, the cell culture layer 22 can still be provided at a slight angle relative to the cell culture layer-receiving surface 34 of a cell culture plate 32. Thus, when referring to a sloping cell culture layer, it is meant that the cell culture layer 22 can be configured so as to be provided with a sloping angle with respect to the cell culture layer-receiving surface 34 of a cell culture plate 32. When considering the location of the inlet reservoir 24 relative to the outlet reservoir 26, the angle can be such that there is a descending slope from the inlet reservoir 24 to the outlet reservoir 26, or the angle can be such that there is an ascending slope from the inlet reservoir 24 to the outlet reservoir 26. For instance, when a cell culture medium is provided in the inlet reservoir 24 and in the outlet reservoir 26, and a test substance is placed into the outlet reservoir 26 but not in the inlet reservoir 24, it can be beneficial to provide the cell culture layer 22 with a slight descending slope toward the outlet reservoir 26 if it is desired to reduce the flow of the test substance into the inlet reservoir 24. Similarly, when a cell culture medium is provided in the inlet reservoir 24 and in the outlet reservoir 26, and a test substance is placed into the inlet reservoir 24 but not in the outlet reservoir 26, it can be beneficial to provide the cell culture layer 22 with a slight descending slope toward the inlet reservoir 24 if it is desired to reduce the flow of the test substance into the outlet reservoir 26.
In some implementations, still when the inlet reservoir 24 and the outlet reservoir 26 are provided in a longitudinally spaced-apart relationship, one of the compartments of the cell culture layer 22 can include an anti-return feature. An anti-return feature refers to a structural feature that encourages the flow of a fluid in a preferential direction. For instance, when a cell culture medium is provided in the inlet reservoir 24 and in the outlet reservoir 26, and a test substance is inserted into the outlet reservoir 26 but not in the inlet reservoir 24, it can be beneficial to provide an anti-return feature in proximity of the outlet reservoir 26, for instance in proximity of a transition between the channel 28 and the outlet reservoir 26, to reduce backflow of the test substance into the inlet reservoir 24 while maintaining fluid communication between the inlet reservoir 24 and the outlet reservoir 26. Similarly, when a cell culture medium is provided in the inlet reservoir 24 and in the outlet reservoir 26, and a test substance is inserted into the inlet reservoir 24 but not in the outlet reservoir 26, it can be beneficial to provide an anti-return feature in proximity of the inlet reservoir 24, for instance in proximity of a transition between the channel 28 and the inlet reservoir 24, to reduce flow of the test substance into the outlet reservoir 26 while maintaining fluid communication between the inlet reservoir 24 and the outlet reservoir 26.
Referring to
Still referring to
The configuration of the inlet reservoir 24 or the outlet reservoir 26 can be chosen according to the type of biological material that is intended to be received therein. For instance, an inlet reservoir 24 that is configured to receive elongating organoids such as neurospheres, neuro-organoids, or any other type of biological material containing neurons, therein can be chosen to include the downwardly converging portion as described above. In such implementations, the downwardly converging portion of the inlet reservoir 24 can contribute to stabilize the neurospheres or the neuro-organoids against the converging walls of the inlet reservoir 24 and to direct axons toward the channel 28.
In some implementations, the inlet reservoir 24 and/or the outlet reservoir 26 can have a depth, along the z-axis, ranging from about 0.1 mm to about 50 mm. In some implementations, the inlet reservoir 24 and/or the outlet reservoir 26 can have a width, along the x-axis, ranging from between about 0.1 mm to about 200 mm. The width of the inlet reservoir 24 and the outlet reservoir 26 can vary depending on the number of channels 28 that extends therebetween, among other factors. In some implementations, the inlet reservoir 24 and/or the outlet reservoir 26 can have a length, along the y-axis, ranging from between about 0.1 mm to about 200 mm. It is to be noted that the dimensions given above are examples only, and that various sizes are also possible, for instance depending on the intended use of the cell culture device 20.
The inlet reservoir 24 and the outlet reservoir 26 are in fluid communication via at least one channel 28 extending therebetween. The number of channels 28, or the density of the channels 28, between the inlet reservoir(s) 24 and outlet reservoir(s) 26 can vary. For instance, in some implementations, there can be anywhere from 1 to 100 channels between a pair of an inlet reservoir 24 and an outlet reservoir 26, or there can be more than 100 channels between a pair of an inlet reservoir 24 and an outlet reservoir 26. The distribution of the channels 28 can vary along the width of the cell culture layer 22 and thus of the inlet reservoir 24 or outlet reservoir 26, the width of the cell culture layer 22 being considered as extending along the x-axis in the Figures. For instance, the channels 28 can be provided at a distance between each other that is relatively constant, such as illustrated in
The channels 28 of the cell culture layer 22 are configured to at least enable orienting an elongated component of a biological material away from the inlet reservoir 24. As used herein, the expression “elongated component” of a biological material refers to any type of biological structure or cell type that typically grows in an elongated fashion, such as hairs or axons, for instance. The channels 28 described herein provide a stabilizing environment for such elongated component of a biological material to grow or to be maintained in an elongated configuration, which can contribute to preventing entanglement and promote a directional growth of the elongated component.
In some implementations, the channel 28 can be configured with dimensions and a structure that enables maintaining the biological material or a given component of the biological material in a given position, which can enable avoiding or reducing the movement of the biological material or the given component of the biological material along the x-axis, the y-axis and/or the z-axis. Maintaining a given component of the biological material or the biological material in a given position can also contribute to avoiding or reducing floating of the biological material in the liquid medium and keeping it immersed in a specific solution or medium, and/or avoiding or reducing movement of the biological material or of a component of the biological material from one compartment to another, and/or avoiding or reducing movement of the biological material towards one or the other reservoir, and or to the sides. The channel 28 can also contain an additional structure to lock the biological material or a component of the biological material in place to orient the direction of the biological material or the component of the biological material from one reservoir or another. For example, the follicles side can be facing one reservoir while the hair coming out of the follicles can be facing another reservoir.
In some implementations, the channel 28 can include, or can consist of, an elongated component receiving portion 30 that is sized and configured for orienting the elongated component of the biological material away from the inlet reservoir 24. For instance, in some implementations when the multicellular component of the biological material is received in the inlet reservoir 24, the channel 28 can include an elongated component receiving portion 30 that is sized and configured for selectively retaining the multicellular component of the biological material upstream of the channel 28, i.e., within the inlet reservoir 24, while the elongated component extends within the channel 28, i.e., within the elongated component receiving portion 30. The elongated component receiving portion 30 can also be configured to constrain or hold the elongated component of the biological material in position, i.e., to reduce the movement of the elongated component of the biological material. For instance, when the cell culture layer 22 is configured to culture hairs and associated follicles, the elongated component receiving portion 30 can be sized and configured to hold the hair in place and prevent it from passing through the channel 28 completely, as hair follicles are thicker at the follicle end and thinner at the hair end.
In some implementations, the channel 28 can further include a multicellular component receiving portion 31 configured for receiving at least a portion of the multicellular component of the biological material therein. When the channel 28 includes a multicellular component receiving portion 31, the elongated component receiving portion 30 is provided downstream of the multicellular component receiving portion 31. In implementations where the multicellular component of the biological material is received in the inlet reservoir 24, the multicellular component receiving portion can be omitted. In some implementations, a portion of the multicellular component of the biological material can be received in the inlet reservoir 24, and another portion of the multicellular component of the biological material can be received in the multicellular component receiving portion 31. When the channel 28 includes a multicellular component receiving portion 31, with an elongated component receiving portion 30 provided downstream of the multicellular component receiving portion 31, the elongated component receiving portion 30 can be sized and configured for orienting the elongated component of the biological material toward the outlet reservoir and for preventing the multicellular component from travelling past the multicellular component receiving portion 31.
It is to be understood that the size and configuration of the channel 28, or of portions of the channel 28, can be adapted to selectively retain a multicellular component of the biological material therein and preventing the multicellular component to travel downwardly past the multicellular component receiving portion 31, and to maintain the elongated component of the biological material in an elongated configuration downstream of the multicellular component receiving portion 31, i.e., in the elongated component receiving portion 30.
One of the objectives of the interaction of the channels 28 with the inlet reservoir 24 is to enable the elongated component of the biological material to be cultured in an organized fashion while another component of the biological material, or another biological material, remains in the inlet reservoir 24 of the cell culture layer 22 or in the multicellular component receiving portion 31 of the channel 28, so any configuration of the channels 28 that can achieve such objective can be suitable.
For instance, when the cell culture layer 22 is configured to culture neurons, the inlet reservoir 24 or the multicellular component receiving portion 31 of the channel 28 can be configured to receive clusters of cell bodies therein, or neurospheroids, neuro-organoids, or any other type of biological material containing neurons, and the channel 28, or the remainder of the channel if the channel includes a multicellular component receiving portion 31, can be configured to receive the axons, the channel 28 comprising an elongated component receiving portion 30 that is sized and configured to maintain the elongated configuration of the axons. Examples of types of neuronal cells that can be used for the preparation of the compartmentalized in vitro model can include mammalian neurons, such as rodent embryonic neurons, and neurons derived from induced pluripotent stem cells, such as human induced pluripotent stem cells, for instance. The option of growing different types of neuronal cells when preparing the compartmentalized in vitro model can increase the versatility of the resulting biological model, which in turn can offer a wider range of opportunities for the various needs of the industry. Using human-derived cells can also be beneficial to provide reproducible and accurate results that can facilitate the translation of the drugs or compounds testing to human studies.
In another example, the cell culture layer 22 can be configured to culture hairs and associated follicles. In such implementations, the inlet reservoir 24 or the multicellular component receiving portion 31 of the channel 28 can be configured to culture the follicles, while the channel 28, or the remainder of the channel 28 if the channel includes a multicellular component receiving portion 31, can be configured to receive the hairs, the channel 28 comprising an elongated component receiving portion 30 that is sized and configured to maintain the elongated configuration of the hairs. In this example, one hair can be received in a given channel 28 of the cell culture layer 22, or alternatively, each channel 28 of the cell culture layer 22 can be configured to receive more than one hair.
In some implementations, the channel 28 includes a converging portion 33 that converges away (tapers inwardly such as shown in
In implementations where the channel 28 includes a multicellular component receiving portion 31, the converging portion 33, if present, can be provided downstream of the multicellular component receiving portion 31. In such implementations, the converging portion 33 can define at least a portion of the elongated component receiving portion 30. Alternatively, at least a portion of the multicellular component receiving portion 31 can form part of the converging portion 33, or be defined by the converging portion 33, of the channel 28. In other words, at least a portion of the multicellular component receiving portion 31 can be defined by a portion of the channel 28 that is converging inwardly toward the outlet reservoir 26 and that is sized and configured to receive a multicellular component of a biological material therein, while the remainder of the converging portion 33 is sized and configured to receive an elongated component of the biological material therein. Accordingly, when the channel 28 comprises a converging portion 33, the converging portion 33 can define at least a portion of the multicellular component receiving portion 31, or the converging portion 33 can define at least a portion of the elongated component receiving portion 30. In some implementations, the converging portion 33 can define both at least a portion of the multicellular component receiving portion 31 and at least a portion of the elongated component receiving portion 30.
In the implementations shown in
In the implementations shown in
The converging portion 33 of the channel 28 can take various configurations. In the implementations shown in
In the examples shown in
In the example shown in
In the example shown in
In some implementations, the converging portion 33 of the channel 28 can be shaped as a cup, the curved surface of the cup forming the converging portion 33 of the channel 28. In such implementations, the curved surface of the cup forming the converging portion 33 can be sized and configured such that a multicellular component of the biological material lightly leans against the curved surface of the cup, to prevent the multicellular component from travelling downstream toward the outlet reservoir 26. Furthermore, in such implementations, the converging portion 33 can be considered as including a multicellular component receiving portion 31 that receives the multicellular component therein, with the remainder of the channel 28, i.e., the elongated component receiving portion 30, is sized and configured to receive an elongated component of the biological material therein.
In some implementations, the converging portion 33 can include a frustoconical converging portion, similar to the example shown in
In some implementations, the converging portion 33 of the channel 28 can include converging sections that are each converges at a corresponding angle toward the outlet reservoir 26.
In some implementations, the channel 28 can have substantially constant width along the x-axis, or can include a constant width portion 44 provided along at least a portion located downstream of the converging portion 33, the constant width portion 44 having a substantially constant width throughout its length, as illustrated for instance in
In some implementations, the transition between successive converging sections of the converging portion 33 can include a curved transition. Alternatively, the transition between successive converging sections of the converging portion 33 can include a sharp transition. For instance, in the implementations, shown in
Referring now more particularly to
In some implementations, and as mentioned above, the channel 28 can include a multicellular component receiving portion 31 that is sized and configured to retain a multicellular component of a biological material therein, which can also be referred to as a non-elongated component of a biological material, upstream of the elongated component receiving portion 30. In other words, the reduction in size of the channel 28 from the multicellular component receiving portion 31 to the elongated component receiving portion 30 can be such that at a certain location along the length, i.e., along the y-axis, of the channel 28, the channel 28 becomes too small for the multicellular component of the biological material or the non-elongated component of the biological material to travel further down along the y-axis of the channel 28. In such implementations, the channel 28 thus includes at least one section that corresponds to the elongated component receiving portion 30 and that has a smaller width or diameter than the width or diameter of the multicellular component of the biological material that will be received upstream of the elongated component receiving portion 30.
For instance, when the biological material is a neurosphere, the cluster of neuronal cell bodies of the neurosphere can be received into a multicellular component receiving portion 31 of a channel 28, and the axons can extend in an elongated component receiving portion 30 of the channel 28 that is sized according to the size of the neurosphere, such that the neurosphere containing the cell bodies is retained upstream of the elongated component receiving portion 30. For example, and with reference to
When the multicellular component receiving portion 31 is shaped as a cup, the size and configuration of the multicellular component receiving portion 31 can be adapted in accordance with the shape of the multicellular component of the biological material or the non-elongated component of the biological material, and can substantially match the shape of the multicellular component of the biological material or the non-elongated component of the biological material. For instance, for a neurosphere that is substantially circular, the multicellular component receiving portion 31 can be sized such that the cluster of neuronal cell bodies of the neurosphere leans lightly against the inwardly converging wall 29 of the converging portion 33, with the axons extending within the elongated component receiving portion 30 provided downstream of the multicellular component receiving portion 31, or downstream of the inwardly converging wall 29.
These configurations of the channel 28 can also be implemented for instance when a first biological material is a follicle, and a second biological material is a hair as an elongated component. In such implementation, the multicellular component receiving portion 31 can be sized and configured such that the follicle is retained within the multicellular component receiving portion 31, with the hair extending further into the channel 28 downstream of the multicellular component receiving portion 31. In this example, the follicle would thus be considered as a multicellular component of a biological material (or non-elongated), and the hair shaft would be considered an elongated component of the biological material. A hair shaft can typically have a diameter of 50 microns to 150 microns, and a follicle can typically have a diameter of about 200 microns, such that an elongated component receiving portion 30 having at least a portion that has a diameter of less than 200 microns can enable retaining the hair shaft therein and maintaining it in an elongated configuration, while the follicle is retained upstream in the multicellular component receiving portion 31. This type of configuration of the channel 28 can prevent the hair shaft from travelling down the channel 28, as the follicle retains it via its interaction with the multicellular component receiving portion 31.
When the converging portion 33 includes a succession of converging walls or a continuous converging wall, at least a section of the converging portion 33 can be sized as a multicellular component receiving portion 31 to retain a multicellular component of a biological material or a non-elongated component of a biological material upstream of the elongated component receiving portion 30 of the channel 28.
It is to be noted that although the configurations of the channels 28 shown in
In some implementations, the channel 28 can have a substantially constant width, or diameter, along its length, i.e., between the inlet reservoir 24 and the outlet reservoir 26. In such implementations, the channel 28 can be sized for orienting the elongated component of the biological material toward the outlet reservoir 26 and for contributing to maintaining the elongated component of the biological material in position, or stabilized, during culture. For instance, a channel 28 having a substantially constant width between the inlet reservoir 24 and the outlet reservoir 26 can be implemented when the biological material itself has a converging shape, or when the multicellular component of the biological material has a larger size compared to the size of the elongated component of the biological material. An example of such biological material is a hair shaft and associated follicle. In this example, the follicle would thus be considered as a multicellular component of a biological material (or non-elongated), and the hair shaft would be considered an elongated component of the biological material. In such implementations, the channel 28 can have a sufficiently small width to prevent the follicle from entering into the channel 28, such that the follicle remains in the inlet reservoir 24 and the hair grows into the channel 28. For instance, as a hair shaft can typically have a diameter of 50 microns to 150 microns, and a follicle can typically have a diameter of about 200 microns, the channel 28 can have a constant width of less than 200 microns to enable retaining the hair shaft therein and maintaining it in an elongated configuration, while the follicle is retained in the inlet reservoir 24. This type of configuration of the channel 28 can prevent the hair shaft from travelling down into the channel 28. Other examples of biological material having a converging shape also include neurospheres or neuro-organoids. In such implementations, the channel 28 can be sized according to the size of the neurosphere, i.e., such that the cluster of cells of the neurosphere forming the multicellular component of the biological material can be retained in the inlet reservoir 24, and the axons can extend within corresponding channels as the elongated component of the biological material.
The channels 28 of the cell culture layer 22 can be carved, stamped or molded into the cell culture layer 24. In some implementations, the cell culture layer 22 includes channels 28 that are open-top, i.e., that are open to the atmosphere unless a cover is deposited onto the cell culture layer 22. In such implementations, it is thus meant that the top wall 38 of the channels 28 as described above is omitted. When the top wall 38 of the channels 28 is omitted, the converging portion 33 of the channels 28 can be achieved by the side walls 40 converging along the x-axis. As mentioned above, the channels 28 can include a back wall, or the bottom wall 34 of the cell culture plate 32 can act as a back wall, or an additional layer or membrane can be provided underneath the bottom surface of the cell culture layer 22, i.e., between the top surface of the bottom wall 34 of the cell culture plate 32 and the cell culture layer 22. The membrane can include for instance a biological material, or can be made of a biologically inert material.
With reference to
In some implementations, the cell culture layer 22 can further include an inlet well 48 in fluid communication with the inlet reservoir 24, and/or an outlet well 50 in fluid communication with the outlet reservoir 26. The inlet well 48 is configured to receive an inlet fluid medium therein, and the outlet well 50 is configured to receive an outlet fluid medium therein, which can be the same or different than the inlet fluid medium.
The fluid communication between the inlet well 48 and the inlet reservoir 24, or between the outlet well 50 and the outlet reservoir 26, can be established in various ways. In the implementation illustrated in
Similarly, still in the implementation illustrated in
Turning to
Either one of channels of the plurality of inlet reservoir channels 54, the plurality of outlet reservoir channels 58, the inlet reservoir channel 60 and the outlet reservoir channel 62 can include at least one turbulence reducing feature. A turbulence reducing feature can include a step change as described above in reference to the converging of the channel 28 toward the inlet reservoir 24 or the outlet reservoir 26. For instance,
In some implementations, an inlet fluid medium feeding system can be provided to supply an inlet fluid to the inlet well 48, and/or an outlet fluid medium feeding system can be provided to supply an outlet fluid to the outlet well 50. In some implementations, the inlet fluid medium feeding system and the outlet fluid medium feeding system can be combined and provided as a fluid medium feeding system, supplying both an inlet fluid to the inlet well 48, and an outlet fluid to the outlet well 50. The inlet fluid medium feeding system, outlet fluid medium feeding system or fluid medium feeding system can be any suitable structure configured to direct the introduction of a fluid medium into a corresponding well of the cell culture layer 22, and optionally contain a certain volume of fluid medium therein. In some implementations, the inlet fluid medium feeding system, outlet fluid medium feeding system or fluid medium feeding system can include a tube in fluid communication with a corresponding one of the inlet well 48 or the outlet well 50. In some implementations, the inlet fluid medium feeding system, outlet fluid medium feeding system or fluid medium feeding system can include an automated distribution system configured to provide a fluid medium to a corresponding one of the inlet well 48 or the outlet well 50 at given timepoints.
In some implementations and as mentioned above, a cover 66 can be deposited on a top surface 27 of the cell culture layer 22, such as shown in
In the implementation illustrated in
In some implementations, the biological model can be a three-dimensional skin model. A three-dimensional skin model can enable interaction with the elongated component of the biological material that is received in the channels 28, such as axons or hair, for instance. In some implementations, for instance when the biological model includes skin cells, the cover 66 can also include a collagen membrane. The cover 66 can also be made of any suitable biological tissue such as the intestines, muscles, the cornea, tumors, the heart, etc. As noted above, the biological model can also be cultured cells of such biological tissues. It is noted that the biological model can also be referred to as a three-dimensional biological tissue model.
In some implementations, when the biological tissue is a three-dimensional skin model, the three-dimensional skin model can include various types of cells, and generally include keratinocytes, Merkel cells, Langerhans cells, and melanocytes. The three-dimensional skin model can be a scaffold-based 3D model, which can reproduce the mechanical structure and the functionally of primary biological tissue. In scaffold-based 3D models, cells are grown on a support scaffold. The support scaffold can be made of natural polymers, such as collagen, fibronectin, elastin, fibrin, silk, alginate, chitosan, fibrin, or GAGs. The support scaffold can also be made of synthetic polymers, such as poly(ε-caprolactone) (PCL), polylactic acid, polyglycolic acid, polylactic-co-glycolic acid (PLGA), polyhydroxybutyrate, and polyethers such as polyethylene glycol (PEG) or PEG co-polymers.
In some implementations, when the cover 66 is made of a biologically inert material, the cover 66 can be made of various materials such as glass or plastics. Examples of plastics include PS, PP, COP, COC, and PC, to name a few. The cover 66 can have for instance a thickness ranging from 0.04 mm to 5 mm, although other thicknesses are also possible depending on the intended use. The thickness of the cover 66 can be determined so as to facilitate gas exchange with cell culture incubator environments. The cover 66 can also be made of a porous material like a sieve, a microporous membrane, or a porous microfiber, which can also contribute to facilitate gas exchange with cell culture incubator environments.
The cover 66 can be configured to fully cover the openings on the cell culture layer 22 such as the inlet reservoir 24, the outlet reservoir 26, and the inlet well 48 and the outlet well 50 if present. Alternatively, the cover 66 can partially cover these openings. For example, the cover 66 can be configured to cover the inlet reservoir 24 and the outlet reservoir 26, while the inlet well 48 and/or the outlet well 50 can remain open to atmosphere. The above-mentioned scenarios are examples only and as such, the cover 66 can have any configuration that enables desired receptacles of the cell culture layer 22 to be covered or to remain open to atmosphere.
In the implementation shown in
In some implementations, the cell culture device 20 can be configured to be used in combination with, or can include, an electrode or a group of electrodes such that the electrode or group of electrodes can be in contact, either direct or indirect, i.e., in electrical communication, with the biological material present in at least one of the inlet reservoir 24, the channels 28 and the outlet reservoir 26. The electrode or group of electrodes can take the form of an electrode layer that can be placed underneath the cell culture layer 22, or on the top surface 27 of the cell culture layer 22. The electrode layer can be in direct contact with the cell culture layer 22, or can be in indirect contact with the cell culture layer 22, for instance if a membrane is placed underneath the cell culture layer 22 and the electrode layer is placed between the membrane and the bottom wall 34 of the cell culture plate 20, or if a cover 66 is placed on the top surface 27 of the cell culture layer 22 and the electrode layer is received on top of the cover 66. In yet other implementations, the electrode layer can form part of the biological model such that the biological model grows around it. It is to be understood that any suitable sequence of the cell culture layer 22, membrane, cover and electrode layer can be implemented depending on the in vitro compartmentalized model that is desired to be obtained.
In some implementations, multiple electrode layers can be provided according to a combination of any of the locations described above, e.g., under the cell culture layer, under the biological model, within the biological model, or above the biological model.
It is to be understood that the electrode layer can be provided in proximity of the channels 28 of the cell culture layer 22 and/or of the biological model, either directly or indirectly in contact therewith. The proximity of the electrode layer with the biological material being cultured in the cell culture layer 22 or the cells of the biological model can contribute to improving the detection and stimulation of the cells. When the electrode layer is provided in proximity of the cells, the distance between the electrode(s) of the electrode layer and the cells can be in the range of micrometers or millimeters, for instance.
In some implementations, the electrode can be configured to provide an electrical signal to stimulate cells growing in the inlet reservoir 24, in the channels 28 and/or in the outlet reservoir 26, or the cells of the biological model. The electrode can also be configured to collect, and/or record, and/or measure, and/or detect the response of cells to stimulation. In some implementations, the same electrode can be configurable to sequentially perform different actions. For instance, the electrode can be configured to collect a signal at a given timepoint, and at a subsequent timepoint, the electrode can be configured to provide an electrical signal. In some implementations, the electrode can be configured to detect an optical signal or an electrical signal.
In some implementations, the distribution of the electrodes over the surface area of the electrode layer can be such that it corresponds at least partially to the spatial distribution of the channels 28 of the cell culture layer 22, or vice versa, instead of the electrodes being provided randomly relative to the spatial distribution of the channels 28 of the cell culture layer 22. Providing the electrodes in such a configuration can enable obtaining electrodes in an organized fashion which in turn, can enable to better target the function of the electrodes over a controlled architecture and/or number of cells that are growing in the inlet reservoir 24, the channels 28, and/or the outlet reservoir 26. For instance, when neurons are cultured using the cell culture device as described herein, with cell bodies being received in the inlet reservoir or in the channel and axons extending within the channels, electrodes that are provided according to a controlled architecture determined at least in part according to the configuration of the channels can contribute to improve the stimulation of the neuronal cells or the detection of a signal from the neuronal cells.
In some implementations, the electrode can comprise at least one metallic electrode, at least one metal oxide electrode, at least one carbon electrode, a multi-electrode array, and/or at least one field effect transistor detectors.
In some implementations, the cell culture device can include any other types of sensors that can stimulate cells or measure responses of cells to stimulation. Examples of sensors can include optical sensors, chemical sensors, and electrical sensors, for instance.
In some implementations, the cell culture device can include an electrode set provided proximate to a biological material, which can be for instance the biological material cultured in the inlet reservoir, the channels and/or the outlet reservoir, and/or the biological model used in combination with the cell culture layer. The electrode set can include at least one electrode configured to collect an electric signal associated with at least a portion of the biological material. The electrode set can take the form of an electrode layer as described above, or can take a different form. The electrode set can include more or more electrodes. The electrodes can enable providing electrical read-outs comprising one or more of potential recordings, impedance spectroscopy, voltammetry and amperometry.
In some implementations, the cell culture device can include an electronic device in ohmic connection with the electrode described above. The electronic device can include for instance a sensing device or a stimulating device, and can be configured for providing electrical read-outs comprising one or more of potential recordings, impedance spectroscopy, voltammetry and amperometry. The electronic device can be located within a cell culture plate into which is received the cell culture layer, or be provided in proximity thereof.
In some implementations, when the biological material includes neuronal cells, the cell culture device can include a sensor configured for stimulating the neuronal cells, measuring a response from the neuronal cells to stimulation, providing an outlet and/or receiving an inlet. The sensor can include for instance an optical or an electrical transducer.
The general steps of a method for preparing a compartmentalized in vitro model using a cell culture layer received in a cell culture plate will now be described in further detail.
The method detailed in the following paragraphs can be implemented using a cell culture device 20 as described herein, of which examples are shown in
In some implementations, the method for preparing a compartmentalized in vitro model can be used to model a biological material that includes a multicellular component that is a non-elongated component, and an elongated component. In the context of the present description, the use of the expression “non-elongated” and of the term “elongated” can be interpreted as relative terms when such components are compared to one another. For instance, when the biological material is a neurosphere, the cluster of neuronal cell bodies can be considered as a non-elongated multicellular component of the biological material, and the axons would be considered as elongated components of the biological material. Thus, the multicellular component of the biological material can have an aspect ratio that is different than 1:1, but the multicellular component is generally less elongated than the elongated component and therefore is considered as a non-elongated component. The differentiation between the non-elongated component and the elongated component of the biological material can also be made in relation to the size of the channel into which the elongated component of the biological material is intended to be inserted. For instance, in the case of a neurosphere as a biological material, the channels can be configured so as to prevent the cluster of neuronal cell bodies to enter the channels or the elongated component receiving portion of the channel, and can rather be sized to selectively enable the axons to enter the channel or the elongated component receiving portion of the channel, resulting in the axons being considered as the elongated component and the cluster of neuronal cell bodies as the non-elongated component.
In some implementations, the method for preparing a compartmentalized in vitro model can be used to model a first biological material having a multicellular component, and a second biological material, the second biological material comprising an elongated component biologically interacting with the first biological material. In this scenario, the first biological material can be for instance follicles, and the second biological material can comprise hair. As mentioned above, the reference to an “elongated component” can be a relative term to compare the configuration of the elongated component versus the configuration of the first biological material, that is less elongated than the elongated component of the second biological material. The differentiation of the first biological material and the elongated component of the second biological material can also be made in relation to the size of the channel into which the elongated component of the second biological material is intended to be inserted. In other words, the channel or the elongated component receiving portion of the channel can be generally configured to limit the entry of the first biological material therein, but can be sized to receive the elongated component of the second biological material therein.
The method can include supplying a fluid culture medium to an inlet reservoir of the cell culture layer. When the cell culture device includes an inlet well and a manifold, or an inlet well and an inlet reservoir channel, the fluid culture medium can be supplied to the inlet well to successively travel from the inlet well to the inlet reservoir.
The multicellular component of the biological material can then be positioned within the inlet reservoir or within the multicellular component receiving portion of the channel of the cell culture layer.
When the multicellular component of the biological material is positioned within the inlet reservoir, at least a portion of the elongated component of the biological material can be inserted, or can grow, into a channel of the cell culture layer that is in fluid communication with the inlet reservoir. For instance, when the biological material includes a neurosphere, the cluster of neuronal cell bodies can be received in the inlet reservoir and/or in a portion of the channel that corresponds to a multicellular component receiving portion, and the axons can then grow into the channel (if the cluster of neuronal cell bodies is received in the inlet reservoir) or the portion of the channel that corresponds to an elongated component receiving portion (if the cluster of neuronal cell bodies is received in the portion of the channel that corresponds to a multicellular component receiving portion).
When at least a portion of the multicellular component of the biological material is positioned within the multicellular component receiving portion of the channel, at least a portion of the elongated component of the biological material can be inserted, or can grow, into the elongated component receiving portion of the channel provided downstream of the multicellular component receiving portion. For instance, when the biological material includes a neurosphere, the cluster of neuronal cell bodies can be received in the multicellular component receiving portion of the channel, and the axons can then grow into the elongated component receiving portion of the channels of the cell culture layer.
It is to be understood that more than one axon can grow into a single elongated component receiving portion, or a single axon can grow into a corresponding elongated component receiving portion.
Alternatively, a first biological material that includes a multicellular component can be positioned in the inlet reservoir of the cell culture layer, and a second biological material can be inserted or can grow into a channel of the cell culture layer that is in fluid communication with the inlet reservoir, the second biological material comprising an elongated component biologically interacting with the first biological material. For example, the first biological material can be follicles as a multicellular component, the follicles being positioned in the inlet reservoir or being received within a multicellular component receiving portion of a channel, and the hairs can be the elongated component of the second biological material that can be inserted or grown into the elongated component receiving portion of the channels of the cell culture layer.
It is to be understood that more than one hair can be inserted into a single elongated component receiving portion, or a single hair can be inserted into a corresponding elongated component receiving portion.
In both scenarios, the channels are configured to maintain the elongated component in a substantially elongated configuration. As described above, the channel can have various features to enable the maintaining of the elongated component in the substantially elongated configuration. For instance, the channel can include a converging portion that converges away from the inlet reservoir, i.e., that converges toward the outlet reservoir when the outlet reservoir is present. The channel can also include a series of inwardly protruding members that can contribute to maintain the elongated member in an elongated configuration within the channel. In some implementations, the series of inwardly protruding members can define arrowhead features that converge toward the outlet reservoir. The channel can include a neck portion. In some implementations, the diameter or width of the channel can be sufficient on its own to maintain the elongated component in the substantially elongated configuration.
The elongated component of the biological material or of the second biological material can extend within the outlet reservoir, when present. The presence of the elongated component in the outlet reservoir can enable contacting the elongated component with a test substance, as will be described in more detail below. In other implementations, the outlet reservoir can be omitted, and the elongated component can terminate within the channel.
When the outlet reservoir is present, the method can include supplying a fluid culture medium to the outlet reservoir of the cell culture layer. When the cell culture device includes an outlet well and a manifold, or an outlet well and an outlet reservoir channel, the fluid culture medium can be supplied to the outlet well to successively travel from the outlet well to the outlet reservoir.
The biological material can be cultured during a certain period of time in the cell culture layer, and subsequently be subjected to testing, or alternatively, the biological material can be subjected to testing without a given period of time dedicated to culturing the biological material. In some implementations, an inlet test substance can be added to the inlet reservoir to perform testing on the non-elongated component of the biological material, or on the first biological material, whichever is present in the inlet reservoir or in the multicellular component receiving portion. In some implementations, an outlet test substance can be added to the outlet reservoir to perform testing on the portion of the elongated component that extends within the outlet reservoir or within the elongated component receiving portion, or on the additional biological material that is present in the outlet reservoir or within the elongated component receiving portion and that is biologically interacting with the elongated component present in the channel.
The inlet test substance and the outlet test substance can be the same or different. The configuration of the cell culture layer as described herein can advantageously enable to produce compartmentalized in vitro models and to test distinct substances on given portions of a biological material or on given biological materials if more than one is present.
For instance, when the first biological material includes follicles and the elongated component of the second biological material includes hairs, the presence of the inlet reservoir can enable adding an inlet test substance to the inlet reservoir, contacting the follicles with the inlet test substance, and subsequently analyzing the response of the follicles to that inlet test substance. As at least a portion of the hairs are extending within the outlet reservoir, an outlet test substance can be added to the outlet reservoir to contact the at least a portion of the hairs, and the response of the hairs to that outlet test substance can subsequently be analyzed. When the follicles and the hairs are each contacted with their respective test substance, the interaction of the follicles and the hairs when contacted with their respective test substance can also be analyzed.
In accordance with another example, a multicellular component of a biological material such as a neurosphere can be placed in an inlet reservoir, an elongated component of the biological material, such as axons of neurons, can grow into the channel, and an additional biological material can be received within an outlet reservoir, and the presence of the inlet reservoir can enable adding an inlet test substance to the inlet reservoir, contacting the multicellular component with the inlet test substance, and subsequently analyzing the response of the multicellular component to that inlet test substance. An outlet test substance can be added to the outlet reservoir to contact the additional biological material, and the response of the additional biological material to that outlet test substance can subsequently be analyzed. When the multicellular component of the biological material and the additional biological material are each contacted with their respective test substance, the interaction of the multicellular component of the biological material and the additional biological material when contacted with their respective test substance can also be analyzed.
The compartmentalized in vitro models that can be obtained according to the techniques described herein can result in in vitro models having a tridimensional structural organization that more closely resemble the ones observed physiologically, which in turn can contribute to improving disease modelling, efficacy and toxicology testing, providing more accurate and reproducible physiologic responses to the substances being tested, and facilitating producing compartmentalized in vitro models that are more easily reproducible from batch-to-batch preparations.
The cell culture device as described herein can enable testing of substances with different viscosity such as oils, creams and cell medium, different solubility, such as oil and water-based compounds in one or both compartments.
Several alternative implementations and examples have been described and illustrated herein. The implementations of the technology described above are intended to be exemplary only. A person of ordinary skill in the art would appreciate the features of the individual implementations, and the possible combinations and variations of the components. A person of ordinary skill in the art would further appreciate that any of the implementations could be provided in any combination with the other implementations disclosed herein. It is understood that the technology may be embodied in other specific forms without departing from the central characteristics thereof. The present implementations and examples, therefore, are to be considered in all respects as illustrative and not restrictive, and the technology is not to be limited to the details given herein. Accordingly, while the specific implementations have been illustrated and described, numerous modifications come to mind.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050967 | 6/16/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63212308 | Jun 2021 | US |
