WO2017213529A1

WO2017213529A1 - A method for manufacturing a cell-culture substrate, a device for the perfusion cell cultures, a method for maintaining cell cultures and a set

Info

Publication number: WO2017213529A1
Application number: PCT/PL2017/000063
Authority: WO
Inventors: Jakub MIELECZAREK; Grzegorz GAZDOWICZ
Original assignee: Uniwersytet Jagielloński
Priority date: 2016-06-10
Filing date: 2017-06-09
Publication date: 2017-12-14
Also published as: PL238379B1; PL417527A1

Abstract

A method for manufacturing the cell-culture substrate (5) from a biocompatible material, for perfusive cell cultures with three-dimensional geometry, in which the material of the substrate solidifies around the matrix (13), which is subsequently removed to obtain a passable culture-medium-supplying channel (6), or set of channels (6), with the previously designed three-dimensional structure. A device for perfusion cell cultures with three-dimensional geometry, containing a mainframe, a system of the cell-culture medium supply and a cell- culture substrate, in which the substrate is manufactured using the method according to the invention. A method for maintaining cell cultures with the use of the device according to the invention, and the set containing the elements of the device and the ingredients necessary to manufacture the cell-culture substrate (5).

Description

A method for manufacturing a cell-culture substrate, a device for the perfusion cell cultures, a method for maintaining cell cultures and a set

The subject of the invention is a method for manufacturing a cell-culture substrate, a device for the perfusion cell cultures, a method for maintaining cell cultures and a set.

Cell cultures rely in keeping cells alive outside a living organism (in vitro) longer than 24 hours. There are invaluable benefits of maintaining cell cultures in laboratories carrying out broadly understood biological research. Firstly, the cell culture is a convenient source of a specific population of cells being the subject of research, such as genetic, biochemical and cytometric or sophisticated microscopic observations. Another benefit of the in vitro culturing is the possibility of precise control of all such parameters as the concentration of substances, pH or temperature. The research results are often determined by the condition of cells, which is a consequence of the appropriate choice of culturing conditions. Observations of cells subject to the action of a certain factor often have to be long-lasting, which requires keeping cells alive during the whole time of an experiment, namely maintaining a culture. As results of observations and measurements performed on living cells very valuable data are obtained, which allows to describe the responses of a cell for different stimuli and therefore the hints on the phenomena that occur in its interior. Cells in a culture are a laboratory model of a living system, which allows to study complexity of the organic matter and also the basis of its functioning on deeper and deeper levels. A significant benefit that is brought by working with cell cultures is a possibility of verification of hypotheses by the study of cells under the action of specific substances, which we suspect of having the biological effect: toxic, healing or signaling. The methodology of cell cultures is also useful in the process of clinical research as well as in studies associated with the regenerative medicine. The most obvious condition for maintaining living cells in a laboratory is providing them with an environment that resembles natural conditions, namely the appropriate temperature, pH, osmolarity, as well as satisfying their basic physiological needs, such as nutrition and gas exchange. To fulfill these conditions the cells are placed in the purpose-designed solutions, so-called media. Additionally, for the majority of time the cell culture is put inside a chamber of the special device, i.e. a so-called incubator, keeping the appropriate temperature as well as optimal values of partial pressures of the respiratory gases. Another condition for maintaining a successful cell culture is a suitable material to which the cells can adhere. Adherence, i.e. a strong mechanical bond with the surrounding environment, is a crucial feature of cells of multicellular organisms, which ensures their stability. A cell dies if it is deprived of a mechanical and chemical signal of the presence of a substance that could be its supporting substrate. Therefore, providing a biocompatible cell-culture substrate is an important issue. An exception are cancerous cells, which can be cultured for a long time in a suspension. One more important aspect is providing the aseptic processing, which protects the culture against an infection by external microorganisms.

Nowadays cell cultures can be prepared in two ways: as the source of cells for a culture one can use fragments of tissue that are freshly extracted from a given organism - so-called explants, or cell lines - the cells that have already been cultured in a laboratory. Explants are placed directly on a culture substrate or are treated by enzymes, which creates a cell suspension. In the case of cell lines, cells after thawing also form a suspension. Cells from the suspension are subsequently sown on a substrate, a layer of the appropriate medium is poured over them, and the whole container is inserted into an incubator. The medium has to be replaced every few days due to the depletion of nutritients and accumulation of by-products of cell metabolism. In each of the discussed cases cells first flatten out, then they reproduce and gradually cover the bigger and bigger surface of the substrate. In all of the discussed cases we ultimately obtain a single layer of cells, covering the whole available surface of the substrate, i.e. a so-called monolayer. Due to occurence of the phenomenon of contact inhibition, cells quit dividing after filling up the whole surface. An exception is again given by the cancerous cells, which form multilayer colonies, although their size is limited by diffusion of nutritients and oxygen to the deepest layers. Nowadays cell cultures are usually maintained in laboratory flasks, on Petri dishes or multiwell plates. Such containers are made from a material that itself may be a biocompatible substrate and allow to observe the culture with the use of an optical microscope.

The commonly studied cell cultures have two-dimensional geometry. The state of a cell in such a monolayer differs from the physiological state. Metabolism of cells in a culture is less efficient and may diverge from the characteristic metabolism of the initial tissue. Breathing occurs mainly via glycolysis. For these reasons, 2D cell culture does not have a sufficient value as the physiological model of a tissue. Single-layer in vitro cultures are a valuable research tool, giving us some idea about functioning of the biological systems, but they are just models of these systems and the results obtained using such a method can not be straightforwardly transferred to the in vivo systems. Different behavior of cells in a culture with respect to cells of a living organism is a consequence of isolating the former from the three-dimensional structure of a tissue. In such cultures there is no specific intercellular interactions, characteristic for the tissue structure - cells do not receive the complete spectrum of stimuli generated by the complex environment of a tissue, organ or the whole organism. The stimuli can be various biochemical signals (concentration of signal substances in the intercellular space or signals directly transmitted from a cell to a cell through the cell junctions) or mechanical stimuli (pressure) and, in the case of excitable cells, also the electric potential of membrane of the neighboring cell. A model system of greater complexity than 2D cultures yet simpler than natural tissues would be a bridge for the further exploration of the functioning of organisms. It would become possible to study not only the properties of cells alone but also completely new properties that arise in the complex intercellular interactions, characteristic for the tissue level. This is exactly the purpose of three-dimensional cell cultures. Functioning of cells in such cultures is more similar to the in vivo situation. Analysis of the behavior of simple systems in which cells form three- dimensional structures leads to interesting studies of cell migration, cellular differentiation and organogenesis, as well as the growth of cancerous tumors or cell responses to different farmaco logical factors, in the future the development of 3D cell culture techniques should create the possibility of production of tissues for clinical applications, e.g. for performing transplantations. The growth of cells in a culture in the third dimension is mainly limited by the necessity to provide every cell with an access to nutritients and especially oxygen. Overcoming this obstruction requires creation of a system of distribution of the above substances, which can follow the example of natural vascular systems.

In the current state of technology there are known various attempts to overcome the above limitations. Trying to solve the problems one creates substrates with different shapes, microcarriers or porous materials.

The patent application US 20090191631 Al (published on 2009-07-30) describes a system with the geometry of a Petri dish, allowing to maintain three- dimensional cell cultures. The proposed system uses a membrane with the„hollow fiber" type structure as a scaffolding for the 3D cell culture. The nutrition of cells is provided by a medium flowing through fibers of the membrane. The membrane fibers are integrated in a microfluidic system, which is connected to an external reservoir. A significant obstruction to wide proliferation of the discussed idea is its constructional complexity, affecting the price of production, and the impossibility of exchange of a substrate.

The patent application US 20110306122 Al (published on 2011-12-15) describes a system for maintaining three-dimensional cell cultures in droplets suspended in the gravitational field. This solution found a commercial implementation as the systems produced by the company Insphero (www.insphero.com). A significant limitation to the above idea is the size of obtained three-dimensional cultures and the lack of possibility of supplying the system with a medium via the process of perfusion.

Microtissues, Inc. (www.microtissues.com) offers mainframes of the size of standard Petri dishes designed for growing three-dimensional cell cultures. They are multi-use systems equipped with matrices having different numbers of wells. Such systems allow to maintain semi-three-dimensional cultures with significantly limited dimensions. However, they do not enable the constant supply of a medium to the culture. Matrices for 3D cell cultures with similar characteristics but the size of standard culture plates are offered by the company 3D Biomatrix (3dbiomatrix.com).

Perfusion systems for cell cultures are produced, among others, by the company MINUCELLS and MINUT1SSUE Vertriebs GmbH (www.minucells.com), as well as Reinnervate Ltd. (reinnervate.com). However, systems included in the offer of these or other similar companies do not give a possibility of perfusion supply for 3D cultures with channels imitating blood vessels.

Despite the previously mentioned studies, patents and implementations devoted to maintaining three-dimensional cell cultures, there is still a need to find an effective solution that would allow cheap and easy manufacturing of such systems, carrying out research on them and keeping alive a large three-dimensional cell aggregate that has the properties arising from the complexity of processes of intercellular signalization, as well as have a system for distribution of nutritients.

The object of this invention is to provide a solution that can be applied to study three-dimensional perfusion cell cultures. The realization of such an object and solution to the problems existing in the prior art, associated with a simple and efficient method of supplying nutritients to the whole volume of the culture substrate, have been achieved in the present invention.

According to the invention, the method for manufacturing a substrate, from a biocompatible material, for perfusion cell cultures with three-dimensional geometry consists in that the material of a substrate solidifies around a matrix, which is subsequently removed, so that one obtains a passable culture-medium-supplying channel, or set of channels, with the previously designed three-dimensional structure.

Preferably, the material of a substrate solidifies in a culture field that has the form of a frame without a bottom and with openings in the walls, which serve as a passage for the supplying hoses to the interior of the culture field.

It is possible to use hoses made from different materials yet it is necessary for them to be sufficiently elastic, so that the system of hoses could preserve its compactness.

Preferably, every channel is connected to a hose.

Preferably, the matrix is composed of thin fibers, in particular, their role can be played by polymer conduits.

Preferably, the matrix is introduced through a hose into the area of manufacturing of the substrate.

Preferably, the frame without a bottom is an element of the mainframe.

Preferably, the frame without a bottom is put on a Petri dish.

Preferably, the frame without a bottom is put on a microscopic slide.

The material of the substrate solidifies in the culture field, insulating the whole system. The matrix present in the culture field leads to the formation of channels, through which there will follow the constant flow of a medium.

Preferably, matrices have the diameter that enables their insertion in the area of manufacturing of the substrate, through the hoses connected to its walls. The matrices, e.g. polymer conduits, are removed after the solidification of the substrate, leaving behind channels for the medium supply. The introduction of conduits necessary for the formation of channels is performed in the state when the supplying system is disconnected.

The device for perfusion cell cultures with three-dimensional geometiy according to the invention comprises a mainframe, a system of the medium supply and a substrate, in which the substrate is manufactured by the method according to the invention.

Preferably, the device according to the invention comprises the mainframe, on which there is a system of hoses and the solidified substrate from a biocompatible material, with channels. In the culture area the mainframe has a notch enabling easy observation of a cell culture with the help of an optical microscope, especially a confocal one.

Preferably, hoses are connected in a configuration that allows the simultaneous supply of a medium to every channel.

Preferably, the mainframe is equipped with handles holding elements of the system.

Preferably, the device according to the invention comprises the elements controlling the flow of a medium through the hoses.

Preferably, the device according to the invention comprises the elements controlling the flow in every channel.

Preferably, the elements controlling the flow of a medium are screw regulators being an integral part of the mainframe. Preferably, the medium-supplying hoses have a symmetry that allows to supply every channel with equal volumes of a medium in a unit of time.

Preferably, the device according to the invention comprises the flow-inducing system.

A flow in the system can be induced via the connection to a (peristaltic or syringe) pump or gravitationaily, by placing the reservoir of a medium above the mainframe.

Preferably, the device according to the invention comprises the outflow for a working substance.

Preferably, the device according to the invention comprises the sensors that allow to control the system operation and biochemical processes occuring in it in real time. Such sensors, e.g. by analysis of the transparency of a medium flowing through the hoses, can be a source of valuable data.

The device according to the invention is distinguished by the characteristic geometry that allows to place the mainframe in a Petri dish with standard dimensions. Therefore, it is compatible with the standard laboratory equipment. in the device according to the invention there is a freedom of creating configurations of connections of a medium-supplying system. In particular, one can consider a configuration with the one-way flow or direction of the flow depending on a channel. The supply system can also be realized in a configuration in which, depending on a channel, one introduces different substances. Such a setup allows e.g. carrying out research devoted to the diffusion of a given agent through the cell culture. Process of the diffusion is there studied via an analysis of ingredients outflowing from a given channel. In this setup the device can find an application in studies of the distribution of pharmaceuticals inside living organisms. The introduction of living cells into the device can be realized in many different ways. In particular, we may list:

1 ) mixing a cell suspension with the fluid material of a substrate (before pouring it over the matrix),

2) introducing cells in the form of a suspension into channels of the prepared system,

3) placing a cell line on the surface of a solidified substrate,

4) a local introduction of cells via an injection to the volume of a solidified substrate. A preferable property of the proposed device is that it can work with different cell-culture substrates. This allows to study a variety of cell lines.

A preferable property of the device according to the invention is also that its construction does not require using advanced production techniques. The skeleton of the construction can be produced (as it happened in the case of the prototype) in the technology of 3D printing. Therefore, the production can be cost-effective even for a small number of copies of the system.

Using the device according to the invention one can easily and quickly form the blocks of agar or other biocompatible material, with the medium-supplying channels. These channels in the substrate and the culture medium flowing through them fulfil the task of supplying nutritients to each of the cells in a culture. In this way the culture is able to grow to a bigger size. Within the presented solution there exists a possibility of obtaining multiple layers of cells, which is impossible to achieve by traditional methods since then the diffusion of nutritients is insufficient to provide the appropriate conditions for all cells.

The device according to the invention can be realized as a modular construction comprising a set of mainframes with different configurations of connections. The device can also be extended by adding the appropriate sensors within every mainframe.

Within the scope of the invention there also falls the method of maintaining cell cultures with the application of the device according to the invention, as well as the set comprising the elements of the device according to the invention and the ingredients necessary to manufacture the cell-culture substrate from a biocompatible material.

Due to the normalized dimensional characteristics, a particularly valuable tool could be gained by research requiring many experimental systems with a high degree of similarity, which are difficult to prepare studying natural three-dimensional cell aggregates.

The subject of the invention is illustrated in the embodiment as presented in a drawing, in which fig. 1 shows a schematic diagram of the device according to the invention, fig. 2 shows the device according to the invention in a top view and an axial section, fig. 3 schematically shows the steps of manufacturing of a cell-culture substrate, fig. 4 schematically shows the steps of filling the device according to the invention with a cell-culture medium, fig. 5 shows the mainframe of the device according to the invention in the version with two channels in a perspective view and a top view, and fig. 6 shows the mainframe of the device according to the invention in the version with four channels in a perspective view and a top view.

The subject of the invention is illustrated in the embodiment as presented in the drawing, in which fig. 3 shows the steps of method realization according to the invention. Fig. 3a and 3b present an insertion of the matrix 13 in the form of a nylon fiber through the silicon hoses 3a into the area prepared for maintaining a cell culture, which is bounded by a plastic frame without a bottom and with openings in the walls, serving as a passage for the supplying hoses to the interior of the culture field. Fig. 3c shows filling of the culture field with the substrate 5 in the form of the 1% solution of agarose hydrogel in the liquid PBS buffer and fig. 3d shows the removal of the nylon fiber 13 after the solidification of the hydrogel 5 that fills up the frame, and the joining of the system of hoses 3 with the connector 4, while the area in which a cell culture is maintained is closed from below by the microscopic slide 14.

According to the next embodiment (fig. 1, fig. 2) of the invention, the substrate manufactured by the method according to the invention is bounded by the frame being an element of the mainframe 1 , while the mainframe is placed on a Petri dish 2, which replaces the microscopic slide 14.

The device for perfusion cell cultures with three-dimensional geometry according to the invention, as shown in the embodiment in fig. 2, on the Petri dish 2 comprises the mainframe 1, in which there is the system of silicon hoses 3 kept by the handles 10 and the solidified agar substrate 5 with two channels 6. The hoses 3 are joined by the plastic connectors 4 in a configuration that enables the simultaneous supply of a medium to every channel 6. The device is equipped with the outflow 9 for a working substance. On the hoses 3 there are the control elements 12 with a clamping screw, allowing to control the flow in a chosen branch of the system of hoses, while the substrate 5 can be monitored through the slit 14, closed by the bottom of the Petri dish.

In another embodiment of the invention the mainframe 1 is produced in the version with additional walls (not shown in the drawing). Then there is no need to place the mainframe on a Petri dish. In such a case the bottom of the culture field is closed by a microscopic slide, enabling the convenient monitoring of the culture. The standard microscopic slide with the thickness of 0.2 mm can be used.

In the embodiment shown in fig. 5 there is presented the mainframe 1 for the device according to the invention in the version with two channels.

In the embodiment shown in fig. 6 there is presented the mainframe 1 for the device according to the invention in the version with four channels.

In both configurations the system is compatible with the standard Petri dishes 2 with the internal diameter of 85 mm. The mainframe 1 of the device according to the invention was made from polylactide (PLA), using the technology of 3D printing. The selected hoses 3 are silicon conduits with the internal diameter of 0.8 mm. The handles 12 are plastic M3 screws with the rounded tips, so that they would not lead to a mechanical damage of the hoses 3. The matrix for forming the channels is made from a nylon string.

As a cell-culture substrate in the performed tests the 1% solution of agarose hydrogel was used in the PBS buffer. Such concentration of hydrogel is commonly applied in the laboratory practice for manufacturing the cell-culture substrates. In order to prepare the material, 100 ml of the PBS buffer was poured into a beaker and placed on a magnetic stirrer with the heating function. 1 g of agarose was added to the buffer when it reached 60°C. After obtaining a homogenous mixture, the process of stirring and heating was stopped. Subsequently, using an automatic pipette, the prepared substance was introduced into the frame that is a central part of the mainframe and in which there is inserted the matrix of channels. The hydrogel solidified and next we realized the procedure of forming and filling up the channels.

The device according to the invention was filled with a medium. Taking into account the surface tension, this procedure had to be performed in the appropriate sequence, by opening the flow separetely in each of the channels. It was possible due to the use of clamping screws (fig. 2, element 12), enabling to stop the flow in a chosen channel. The filling sequence, in the case of a mainframe with two channels, is depicted in fig. 4. In fig. 4a, there is shown the moment in which the flow is blocked in one of the channels by tightening up the clamping screw 12a. This allows to fill up the other channel, where the flow is unobstructed (the clamp 12b is relaxed). In fig. 4b there is shown the moment in which the clamp 12a is relaxed, while using the clamp 12b the flow is stopped in the full channel. In this way the flow is induced in the previously empty channel. When the latter becomes full, the clamp 12a is relaxed, enabling the constant flow of the medium through both channels.

The performed tests included: filling and checking the tightness of connections between the culture field and the supply system, the methodology of forming the channels, and the diffusion of a medium (in the tests we used a pigment, in order to visualize the diffusion process) from the channels to the substrate. The flow in the system was induced by a peristaltic pump or a syringe pump 7.

The tests demonstrated that:

1) The proposed methodology of forming the channels works in practice and allows to obtain a tight connection between a cell culture and the supply system.

2) The system is durable under the long flow of a fluid (i.e. a medium).

3) The observed diffusion of a pigment leads to the conclusion that the system enables the medium supply to the whole volume of a cell culture.

4) There exists a possibility of levelling the flow in all the channels, as it is shown by an analysis of the pigment diffusion process.

Finally, a set in the form of a box was prepared, a set with the bottom that has compartments for respective components of the system: the mainframe, the hoses, the connectors, the ingredients necessary to manufacture a cell-culture substrate, the medium reservoir with the gravitationally-induced flow, and the system manual. The set comprises a complete set of elements that allow to manufacture a substrate for maintaining three-dimensional cell cultures.

List of refference numerals

1 - mainframe

2 - Petri dish

3 - hose

4 - connector - cell-culture substrate

- medium-supplying channel - flow-inducing system

- reservoir of a medium

- outflow for a medium

- handle of the hose

- handle of the connector

- flow-regulating element

- matrix

- microscopic slide, observation slit

Claims

Patent claims

1. A method for manufacturing the cell-culture substrate (5), from a biocompatible material, for the perfusion cell cultures with three-dimensional geometry, in which the material of the substrate (5) solidifies around the matrix (13), which is subsequently removed to obtain a passable culture- medium-supplying channel (6), or set of channels (6), with the previously designed three-dimensional structure.

2. A method according to claim 1 , characterized in that the material of the substrate (5) solidifies in a culture field in the form of a frame without a bottom and with openings in the walls, which enable a passage of the supplying hoses (3) to the interior of the culture field.

3. A method according to claims 1-2, characterized in that a hose (3) is fed to every channel (6).

4. A method according to claims 1-3, characterized in that the matrix (13) is composed of thin fibers.

5. A method according to claims 1-4, characterized in that a matrix (13) is introduced through the hose (3) into the area of manufacturing of the substrate (5).

6. A method according to claims 1-5, characterized in that a frame without a bottom is an element of the mainframe (1).

7. A method according to claims 1-6, characterized in that a frame without a bottom is put on a Petri dish.

8. A method according to claims 1-7, characterized in that a frame without a bottom is put on a microscopic slide.

9. A device for the perfusion cell cultures with three-dimensional geometry, comprising a frame, a system of the cell-culture medium supply and a cell- culture substrate, in which the substrate is manufactured according to claims 1-8.

10. A device according to claim 9, characterized in that comprises the mainframe (1), on which there is a system of hoses (3) and the solidified substrate (5) from a biocompatible material with the channels (6).

11. A device according to claim 10, characterized in that the hoses (3) are connected in a configuration that allows the simultaneous supply of a medium to every channel (6).

12. A device according to claims 10-11, characterized in that the mainframe (1) is equipped with handles (10) holding elements of the system.

13. A device according to claims 10-12, characterized in that the elements (12) are contained that control the flow of a medium through the hoses (3).

14. A device according to claim 13, characterized in that comprises the control elements (12) that control the flow in every channel (6).

15. A device according to claim 14, characterized in that the elements (12) controlling the flow through the hoses (3) are screw regulators being an integral part of the mainframe (1),

16. A device according to claims 10-15, characterized in that the medium- supplying hoses (3) have a symmetry that allows to supply every channel (6) with equal volumes of a medium in a unit of time.

17. A device according to claims 9-16, characterized in that comprises the flow- propelling system (7).

18. A device according to claims 9-17, characterized in that comprises the reservoir (8) of a medium.

19. A device according to claims 9-18, characterized in that comprises the outflow (9) for a working substance.

20. A device according to claims 9-19, characterized in that comprises the sensors that allow to control the system operation and biochemical processes occuring in it in real time.

21. A method for maintaining cell cultures with the use of the device according to claims 9-20.

22. The set comprising the elements of the device according to claims 9-20 and the ingredients necessary to manufacture the substrate (5) from a biocompatible material.