The invention relates generally to modeling myelination in vitro. More particularly, the invention relates to a cell culturing platform, to a cell culture system, and to a method for modeling the myelination in vitro.
Myelin is material that forms layers, i.e. myelin sheaths, around neuronal cell processes, i.e. around axons of neuronal cells. The myelin is essential for the proper functioning of the nervous system. The production of the myelin sheath is called myelination. In humans, the production of myelin begins in the 14th week of foetal development, and thus myelin exists in the brain already at the time of birth. During infancy, myelination occurs quickly and continues through the adolescent stages of life. Myelinated axons are white in appearance, hence the “white matter” of the brains. The fat helps to insulate the axons from electrically charged atoms and molecules. These charged particles are found in the fluid surrounding the entire nervous system. Myelin is also a part of the maturation process leading to child's fast development, including crawling and walking in the first year. Demyelination is the loss of the myelin sheath insulating the nerves, and it is an indicator of some neurodegenerative autoimmune diseases, including multiple sclerosis “MS”, acute disseminated encephalomyelitis, Neuromyelitis Optica, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, central pontine myelinosis, inherited demyelinating diseases such as leukodystrophy, and Charcot-Marie-Tooth disease. Demyelination can also occur after traumatic injury such as spinal cord injury as a secondary degeneration process.
Therapeutic interventions to prevent demyelination, i.e. myelin loss, include neuroprotection based on drugs, enhancement of endogenous myelin formation based on drugs, and replacement of lost cells, i.e. transplantation.
In vitro models of myelination are useful in testing and development of drugs and in development of transplantation therapies. Publication Park J., Koito H., Li J., Han A.: High-throughput compartmentalized CNS neuron culture platform for axon degeneration/regeneration study, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3-7 Oct. 2010, Groningen, The Netherlands, pp. 860-262 presents a cell culturing platform suitable for modelling the myelination in vitro. The cell culturing platform is a multi-compartment microfluidic co-culture platform composed of one soma compartment for neurons and six axon compartments. The soma compartment and axon compartments are connected by arrays of axon-guiding channels that function as physical barriers to confine neuronal somas in the soma compartment, while allowing axons to grow into the axon compartments. The oligodendrocytes are loaded into the axon compartments and they can interact with axons but not with the neuronal somas. In some cases it can be, however, challenging to detect whether or not the myelination has taken place in the axon compartments.
The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying and non-limiting embodiments of the invention.
In accordance with the invention, there is provided a new cell culturing platform suitable for culturing e.g. neuronal cells and myelinating cells so as to model myelination in vitro. The term “myelination” includes at least the following processes: 1) myelination, 2) demyelination and 3) remyelination which can all be modeled in this new culturing platform. A cell culturing platform according to the invention comprises solid material adapted to constitute:
The one or more first channels are suitable for guiding growth of neuronal cell processes of the neuronal cells from the one or more first cell chambers to the process chamber and for inhibiting the somas of the neuronal cells from moving from the one or more first cell chambers to the process chamber. The process chamber constitutes a room for the myelination of the neuronal cell processes by myelinating cell processes of the myelinating cells. The above-mentioned second channels are suitable for guiding growth of the myelinating cell processes of the myelinating cells from the second cell chamber to the process chamber and inhibiting the somas of the myelinating cells from moving from the second cell chamber to the process chamber. Hence, the process chamber, where the myelination takes place, is substantially free from the cell somas and thus the detection of the myelination is facilitated. The process chamber has an elongated shape so that each of the one or more first channels is connected to one of shorter sides of process chamber and each of the one or more second channels is connected to one of longer sides of the process chamber, the longer sides of the process channel being at least three times longer than the shorter sides of the process channel.
In accordance with the invention, there is provided also a new cell culture system for modeling myelination in vitro. A cell culture system according to the invention comprises a cell culturing platform according to the invention, wherein:
In accordance with the invention, there is provided also a new method for modeling myelination in vitro. A method according to the invention comprises culturing neuronal cells and myelinating cells in a cell culturing platform according to the invention, wherein:
A number of exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims.
Various exemplifying and non-limiting embodiments of the invention both as to constructions and to methods of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.
The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in dependent claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of “a” or “an”, i.e. a singular form, throughout this document does not exclude a plurality.
The exemplifying and non-limiting embodiments of the invention and their advantages are explained in greater detail below with reference to the accompanying drawings, in which:
In a cell culturing platform according to an exemplifying and non-limiting embodiment of the invention, the sum of cross-sectional areas of the second channels is at least two, or preferably at least three, times greater than the sum of cross-sectional areas of the first channels. The cross-sections of the first channels are taken along a plane parallel to the xz-plane of a coordinate system 199, and the cross-sections of the second channels are taken along a plane parallel to the yz-plane of the coordinate system 199. The cross-sections of the first channels may have a rectangular form, or some other suitable form such as e.g. a half circle. Correspondingly, the cross-sections of the second channels may have a rectangular form, or some other suitable form such as e.g. a half circle. The average number of myelinating cell processes per each neuronal cell process is at least in some extent dependent on the ratio of the sum of the cross-sectional areas of the second channels and the sum of the cross-sectional areas of the first channels. There can be for example three or more second channels for each of the first channels, i.e. the number of the second channels can be three or more times greater than the number of the first channels.
In the exemplifying cell culturing platform illustrated in
In a cell culturing platform according to an exemplifying and non-limiting embodiment of the invention, each of the first channels 104a and 104b is at least 50% longer than each of the second channels. The first channels should have a certain minimum length for being able to guide the growth of the neuronal cell processes towards the process chamber 103.
The dimensions of the first cell chambers 102a and 102b shown in
The dimensions of the process chamber 103 shown in
The dimensions of the second cell chamber 105 shown in
The dimensions of the first channels 104a and 104b shown in
The dimensions of the second channel 106 and of the other second channels shown in
A cell culturing platform according to an exemplifying and non-limiting embodiment of the invention is made of transparent material so as to enable optical inspection of the myelination of the neuronal cell processes. Thus, myelination in the process chamber can be detected using optical microscopy techniques. The transparent material can be for example polystyrene or polyvinyl chloride with or without copolymers, polyethylenes, polystyrene-acrylonitrile, polypropylene, polyvinylidine chloride, silicone eleastomers, or similar materials.
A cell culturing platform according to an exemplifying and non-limiting embodiment of the invention comprises electrodes and wirings for transferring electrical signals from and to neuronal cells.
Advantageously, there are electrodes at least in the first cell chambers 102a and 102b. In
A cell culturing platform according to an exemplifying and non-limiting embodiment of the invention comprises a circuitry connected to the wirings and adapted to measure time elapsed between a first moment when an electrical signal appears on a first one of the electrodes and a second moment when a corresponding electrical signal appears on a second one of the electrodes. The above-mentioned circuitry is denoted with a reference number 109 in
In the exemplifying cell culturing platform illustrated in
Furthermore, a cell culture platform according to an exemplifying and non-limiting embodiment of the invention includes drug/medium application inlets in the first cell chambers 102a and 102b, in the second cell chamber 105, and/or in the process chamber 103 that facilitate providing drug/medium changes only to desired/dedicated areas of the cell culture platform.
Cell culturing platforms of the kind described above can be fabricated by using a slightly modified, i.e. different masktype and slower but more accurate process, version of rapid prototyping method which is commonly used in fabrication of Polydimethylsiloxane “PDMS” structures. In this method, the PDMS structure is molded by using an SU-8 mold. SU-8 is a commonly used epoxy-based negative photoresist. It is a very viscous polymer that can be spun or spread over a thickness ranging from below 1 micrometer up to above 300 micrometers and still be processed with standard contact lithography. Thus, the SU-8 mold can be fabricated by using standard lithography methods.
The SU-8 mold is fabricated by spin-coating SU-8 photoresist on top of e.g. silicon wafer, the height of the layer can be controlled by changing the spinning speed or viscosity of used SU-8. SU-8 is then hard baked and exposed to UV-light through a lithography mask. During the exposure, the features in the mask are transferred to the SU-8. SU-8 is then baked again and developed. This process is repeated multiple times as each height in the mold requires its own SU-8 layer.
Once the SU-8 mold is completed, the PDMS is molded in it. The PDMS components are mixed together by using 1:10 curing agent—base polymer ratio and poured onto the mold. The PDMS is then exposed to vacuum in order to remove air bubbles. After the vacuum treatment, the PDMS is baked in e.g. 60 degrees for 10 hours. After the bake, the PDMS is cut out of the mold and the necessary inlets for fluids are punched into it by using punching tools. Before using the PDMS structures, they are exposed to oxygen plasma in order to make them hydrophilic.
In a method according to an exemplifying and non-limiting embodiment of the invention, the neuronal cells comprise neurons and/or neural precursor cells of an animal or a human being.
In a method according to an exemplifying and non-limiting embodiment of the invention, the myelinating cells comprise oligodendrocytes, oligodendrocyte precursor cells, and/or schwann cells of an animal or a human being.
In a method according to an exemplifying and non-limiting embodiment of the invention, the cell culturing platform is made of transparent material, and the method comprises optically inspecting, with the aid of a microscope, the process chamber so as to find out whether the myelinating cell processes of the myelinating cells have myelinated the neuronal cell processes of the neuronal cells.
In a method according to an exemplifying and non-limiting embodiment of the invention, the cell culturing platform comprises electrodes for directing electrical signals to the neuronal cells and for receiving electrical signals from the neuronal cells. The method according to this embodiment of the invention comprises measuring time elapsed between a first moment when an electrical signal appears on a first one of the electrodes and a second moment when a corresponding electrical signal appears on a second one of the electrodes, and comparing the measured time with a reference value so as to find out whether the myelinating cell processes of the myelinating cells have myelinated the neuronal cell processes of the neuronal cells.
1. Materials and Methods
1.1 Cells
Neurons and oligodendrocytes, so called myelinating cells, used in these experimental cases were differentiated from human embryonic stem cell line as described by Skottman 2010. Differentiation was performed according to methods previously published by Lappalainen et al. 2010, Sundberg et al. 2011. For oligodendrocyte differentiation, minor modifications were made to the published method. A list of the reference publications is presented at the end of this section “Experimental example cases”.
1.2 Polydimethylsiloxane “PDMS” Structures
PDMS structures were manufactured using soft lithography rapid prototyping techniques, which allow fast fabrication of prototype devices from PDMS and which are described by Wolfe and Whitesides 2005. The manufacturing process is simple; first a mold is made from SU-8 negative photoresist on top of a silicon wafer using standard photolithography techniques as described by Park et al. 2006. Once the mold is ready the PDMS, Sylgard 184, components are mixed together and poured into the mold. Vacuum is then used to degas the PDMS and once degassed the PDMS is baked in an oven. After baking, the PDMS is peeled off the mold and fluid inlets are punched into it using punching tools. Once the PDMS structure has inlets it is ready to be used.
1.3 Preparation of Microelectrode Arrays “MEA”
In-house MEAs were prepared as described by Ryynänen et al. 2011.
1.4 Preparation of Structures for Cell Culture
PDMS structures were either permanently or reversibly bonded onto glass coverslips or MEA using oxygen plasma treatment. Cell culture surface in the structure was coated with laminin for neurons and with mixture of laminin, collagen IV and nidogen for myelinating cells.
1.5 Cell Culture in Structures
A subset of neurospheres and spheres of myelinating cells at the differentiation age of 8-10 weeks were mechanically dissected into smaller aggregates and approximately 10 aggregates were plated on the cell chambers or on the second cell chamber. Neurons in the cell chambers were fed with medium described by Lappalainen et al. 2010 and myelinating cells in the second cell chamber with medium described by Sundberg et al 2011. Media were changed three times per week.
1.6 Analysis
i) Microscopy and Time-lapse Imaging
The growth and behavior of cells in the structure were investigated by phase contrast imaging and time-lapse imaging.
ii) Immunocytochemical Staining and Fluorescence Microscopy
The presence of neurons and myelinating cells in the culture was assessed by immunocytochemical staining. For this purpose, cells in the structure were fixed with 4% PFA and immunostained with markers specific for neurons and myelinating cells as described by Lappalainen et al, 2010 and Sundberg et al. 2011. Samples were viewed with fluorescence microscopy.
iii) MEA Signaling
Electrical activity of neurons in the structure was measured using a MEA system, i.e. Multi Channel Systems. Measurement and analysis was performed as described by Heikkilä et al. 2009.
2. Experimental Example Case 1
Somas of neurons are restricted to the cell chamber whereas neuronal cell processes are guided via first channels to the process chamber:
Neuron aggregates were plated onto the cell chambers of the structure and cultured until analyzed. Neurons were successfully cultured in the cell culturing platform. Somas of the neurons were restricted to the cell chamber and neuronal cell processes were able to enter the first channels and form network in the process chamber.
3. Experimental Example Case 2
Restricted process growth of myelinating cells in the process chamber:
Myelinating cells were plated on the second cell chamber of the cell culturing platform and cultured until analyzed. Myelinating cells were successfully cultured in the second cell chamber and the processes of myelinating cells were able to enter process chamber via the second channels.
4. Experimental Example Case 3
Detection of neuronal activity from the process chamber:
Neuron aggregates were plated onto the cell chambers of the structure and cultured until analyzed. The cell culture platform was inserted on top of in house made MEA. The neuronal cell processes were able to enter the first channels and form network in the process chamber on top of MEA electrodes. The spontaneous activity of the formed neuronal network was measured with MEA.
5. List of the Reference Publications
Skottman H. 2009, Derivation and characterization of three new human embryonic stem cell lines in Finland, In Vitro Cell. Dev. Biol.—Animal, 46:206-209.
Heikkilä T, Ylä-Outinen L, Tanskanen J, Lappalainen R, Skottman H, Suuronen R, Mikkonen J, Hyttinen J, Narkilahti S. 2009, Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro. Exp Neurol. 218 (1):109-16.
Lappalainen R., Salomäki M., Ylä-Outinen L., Heikkilä T J., Hyttinen J A K., Pihlajamäki H., Suuronen R., Skottman H., Narkilahti S. 2010 Similarly derived and cultured hESC lines show variation in their developmental potential towards neuronal cells in long-term culture. Regen. Med. 5:749-62.
Park, J. W., Vahidi, B., Taylor, A., Rhee, S. W., Jeon, N. L. Microfluidic culture platform for neuroscience research. Nature protocols, vol. 1, no. 4, 2006, pp. 2128-2136.
Ryynänen Tomi, Kujala Ville, Ylä-Outinen Laura, Korhonen Ismo, Tanskanen Jarno M A, Kauppinen Pasi, Aalto-Setälä Katriina, Hyttinen Jan, Kerkelä Erja, Narkilahti Susanna, Lekkala Jukka. 2011, All-Titanium Microelectrode Array for Field Potential Measurements from Neurons and Cardiomyocytes—a Feasibility Study. Micromachines 2, 394-409.
Sundberg M., Hyysalo A., Skottman H., Shin S., Vemuri M., Suuronen R., Narkilahti S. 2011, A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production. Regenerative Medicine. 6 (4):449-60.
Wolfe, D., Whitesides, G. Rapid prototyping of functional microfabricated devices by soft lithography. Nanolithography and Patterning Techniques in Microelectronics, Woodhead Publishing Limited, 2005, pp. 76-119.
The non-limiting, specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.
Number | Date | Country | Kind |
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20136312 | Dec 2013 | FI | national |
Filing Document | Filing Date | Country | Kind |
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PCT/FI2014/051017 | 12/17/2014 | WO | 00 |