Patent Application
20210222108
The present invention describes an organoid culture system. Furthermore, the present invention also describes a method for sterilizing a culture system.
Various organoid culture systems, also known as three-dimensional culture systems, have been described in the state of the art, in contrast to the traditional two-dimensional monolayer culture.
Qian, X., et al. described in a scientific article (Cell, 165, 1238-1254) in 2016, the development of a miniaturized spinning bioreactor to generate brain-specific organoids from human induced pluripotent stem cells (iPSCs). These organoids incorporated key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression and a human-specific outer radial glia cell layer. The bioreactor was called SpinZ and fits into a standard 12-well tissue culture plate, drastically reducing media consumption and incubator space. The bioreactor also had a stackable version driven by one common motor, which allowed for comparisons of a large number of conditions in parallel (for protocol optimisation) and reducing the incubator space required. However, the system is not modular nor is it a fully sterilizable system. In addition, SpinZ does not incorporate sensors that allow important parameters, such as cell growth, pH, CO2 and O2 concentration, and temperature, to be measured in real time.
In a scientific article (Stem Cells Translational Medicine (2017), 6, 622-633), Wilkinson, et al. described a scalable method for the generation of self-assembled human lung organoids. The generation of said organoids was achieved by combining collagen-functionalized alginate beads and human fibroblasts in a 4 ml high-aspect ratio vessel (HARV) rotational bioreactor. Moreover, the authors also disclose that a GoPro camera can be mounted onto the HARV to characterize the kinetics of organoid formation. However, said invention does not allow standard culture material to be used, nor does it allow for multiple simultaneous culture. The system is not modular either, and although it allows organoid growth to be characterized with the camera that it incorporates, it does not allow this data to be automatically stored or other physiochemical parameters of interest, such as pH, CO2 and O2 concentration and the temperature, to be monitored and stored.
Patent application WO2015/184273 A1 discloses a bioreactor system for preparing a cardiac organoid. The bioreactor system includes a first vessel having a hollow interior and an open top, in addition to a cannula having a lumen, a porous ring coupled to the cannula, and a balloon catheter. This invention presents a problem of applicability, since the requirement of a balloon catheter limits organoid production to a single cardiac type. Furthermore, it is not a modular bioreactor nor does it allow for simultaneous culture of different tissues and/or different conditions. This bioreactor also does not incorporate the possibility of using standard culture material. Moreover, it does not allow the organoid growth to be monitored in real time or the data related to the culture's physiochemical parameters of interest to be monitored and stored.
Patent application WO2012/104437 A1 discloses a bioreactor for cell culture on a three-dimensional substrate. It is made up of a conical-shaped culture chamber. The bioreactor is used in tissue engineering for producing tissue grafts, in particular a bone or cartilage graft. This bioreactor does not allow for simultaneous multiple culture or the use of standard culture material. Moreover, the bioreactor of said invention does not allow the organoid growth to be monitored in real time or the data related to physiochemical parameters of interest to be monitored and stored.
In view of the aforementioned cited documents, there is evidently a need for an organoid culture system that is modular and sterilizable, and which allows for simultaneous multiple culture and allows cell growth to be monitored in real time, as well as relevant physiochemical parameters, such as pH, CO2 and O2 concentration and temperature, to be monitored and stored. In the present invention, an organoid culture system is described that includes the foregoing features, thus significantly improving the existing systems in the state of the art.
The present invention describes an organoid culture system that allows for simultaneous multiple culture (high-throughput), the reuse of standard culture material and equipment, and real-time monitoring of organoid growth and relevant physiochemical parameters. Moreover, the system is modular and one module can be sterilized independently of another.
One aspect of the present invention relates to a sterilizable organoid culture system comprising:
a culture module comprising:
Therefore, the present invention describes a system that has numerous advantages over other organoid culture bioreactors described in the state of the art. The bioreactor of the present invention allows for multiple simultaneous culture, which allows experiments with different conditions to be conducted in parallel. Moreover, the present invention comprises a modular system, whereby different modules of the system can be coupled and decoupled. Therefore, firstly, it enables the culture module to be sterilized in its entirety, without the need to sterilize the parts of the culture module separately, this being very convenient for keeping the entire culture sterile. In other words, the culture module, which comprises one or more sample wells, can be removed as a block, without the need to disassemble the components thereof.
The present invention also describes a method for sterilizing an organoid culture system comprising:
providing a system like the one defined above in a stand-by configuration such that the stirring module and the control module are decoupled from the culture module;
removing the culture module;
and subjecting the culture module to sterilization.
a culture module (2) comprising:
The present invention describes an organoid culture system and a method for sterilizing an organoid culture system.
With reference to
a culture module (2) comprising:
The basis of the culture module (2) is the stirring of the culture by air bubbles, which also help to facilitate the gas exchange with the culture medium.
In an embodiment of the invention, the controller (84) is configured so that the pressure reading provided by the pressure sensor (83) corresponds to the pressure transfer/time curve selected for each experiment.
In an embodiment of the invention illustrated in
In an embodiment of the invention illustrated in
According to an embodiment of the invention, the air compressor system (80) collects the air from the incubator where the system of the invention is located and drives it towards the nozzles (82), previously passing through the filter (85), that avoids contaminating the culture medium.
As illustrated in
In an embodiment of the invention, the controller (84) is configured to detect a malfunction corresponding to a clogged nozzle (82), a dirty air filter (81), and both. The control system has an air pump control algorithm that, based on the compressor speed data and the pressure read in the chamber prior to the filter (85), allows clogged nozzles (82) or a dirty filter (85) to be detected.
In an embodiment of the invention illustrated in
The support means (10) allows for the comprehensive manipulation of all the samples wells (4) used for the organoid culture. Preferably, the system described in the present invention can be used in combination with a standard culture plate (104). In a standard culture plate (104), the support means (10) and the sample wells (4) form a single unit.
Thus, in a preferred embodiment, standard culture plates can be used in the organoid culture system described in the present invention, such that the bioreactor is widely applicable and the use thereof will not require special devices for organoid culture, unlike other systems described in the state of the art. Furthermore, the use of standard culture plates allows for simultaneous multiple culture, thus allowing for cell culture under different physiochemical conditions on the same plate, in the same experiment.
In an embodiment of the invention illustrated in
an environmental sensorization module (43) comprising one or more sensors (41);
wherein said one or more sensors (41) are configured to measure culture-related parameters selected from cell growth, CO2 concentration, O2 concentration, pH, temperature, humidity and volatile organic compounds.
According to an embodiment of the invention, the one or more sensors (41) are configured to measure the culture-related parameters in real time.
In addition, the system of the invention allows new sensors to be incorporated to record new information in real time that may be relevant.
In an embodiment of the invention illustrated in
a growth monitoring module (50) comprising:
The organoid growth is monitored in real time by means of a computer vision system, which obtains individual images of each sample well (4) and processes them to calculate the size thereof.
In an embodiment of the invention illustrated in
With the fixed focus lens, the image capturing means (51) are adjusted to the resting plane of the organoids, in order to perform a growth estimation based on a 2D projection of the culture.
With the adjustable focus lens, the image capturing means (51) allow several images of each sample well to be captured, corresponding to different planes of the culture, improving the growth estimation, since it allows for a 3D reconstruction of the organoid.
With light field technology, the image capturing means (51) allow the growth to be seen in different planes without an adjustable lens, since light field technology allows the focal plane of the capture to be modified without the need for adjustable focus lenses.
With multi-camera technology, the image capturing means (51) comprise several cameras that focus on the culture from different angles, allowing for the 3D reconstruction of the same.
In an embodiment of the invention illustrated in
According to different embodiments of the invention, the motorized system (52) comprises a system selected from:
In addition, in accordance with an embodiment of the invention illustrated in
According to an embodiment of the invention, the visual guides (53) are encoded and selected from two-dimensional codes and two-dimensional markers (fiducial markers).
The two-dimensional codes, such as QR, allow any type of alphanumeric information to be stored, encoded as points forming a two-dimensional matrix.
The two-dimensional markers (fiducial markers) are also made up of a two-dimensional matrix of points, but unlike two-dimensional codes, the information stored is predefined and usually corresponds to a sequence number for each unique code in a predetermined dictionary. For example, in the case of AprilTag codes, the 36H11 family allows for 518 different codes.
The two-dimensional markers are often used as indices for automatic positioning or detection of predefined elements.
In an embodiment of the invention, a combination of both types of codes is used:
In each particular embodiment, different types and combinations of codes can be used, placed in different positions on the culture plates (104).
In addition, in accordance with an embodiment of the invention illustrated in
In another embodiment of the invention illustrated in
a control module (3) comprising:
The control module (3) can be connected via wired or wireless connection. The following connection modes are supported, among others:
The control module (3) according to the invention may further comprise a system that provides real-time information on the aforementioned physical parameters: cell growth, CO2 concentration, O2 concentration, pH and temperature.
In an embodiment of the invention illustrated in
In another preferred embodiment, the number of sample wells (4) is an even number.
In another preferred embodiment, the number of sample wells (4) is 6, 8, 12, 24, 48 or 96. Preferably, the number of sample wells corresponds to the number of sample wells of a standard culture plate (104).
According to an embodiment of the invention, the system is adapted to be in an operating configuration and a stand-by configuration,
Another embodiment of the invention relates to a method for sterilizing an organoid culture system comprising:
In an embodiment of the method of the invention, the sterilization to which the culture module (2) is subjected is autoclaving, treatment with hydrogen peroxide or treatment with ionizing radiation.
In another embodiment of the invention illustrated in
The real-time measurement of the aforementioned parameters, such as cell growth, CO2 concentration, O2 concentration, pH and temperature, are very advantageous for determining the correct growth of the organoid under suitable conditions.
With reference to
All the information collected from the control module (3) can be accessed at any time that the system is operating, but it can also be stored for subsequent analysis and/or quality control. The information can be stored locally, preferably on a memory card, and/or remotely, on the cloud using any standard network connection.
The system also preferably includes an alarm protocol that constantly alerts in the event of a system failure or an unexpected alteration of the physical parameters measured by the control module (3).
The culture module (2) is coupled/decoupled from the growth monitoring module (50) as shown in
The culture module (2) was sterilized as follows:
The organoids were developed from two tumor cell lines previously obtained from primary RbloxP/loxP HRasV12 (T653) and cRb−/− HRasV12 (T731) astrocytes in SCID mice. These cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS) at 37° C. and 5% CO2.
To establish the neurosphere culture derived from the primary tumor culture, the T653 and T731 cells were washed in phosphate-buffered saline (PBS) solution, trypsinized and recovered by centrifugation in PBS at 1000 rpm for 5 minutes. The cells were resuspended in the DMEM/F-12, GlutaMAX nutrient mix supplemented with 1× B-27 (50×) and 0.02 μg/ml EGF (Human Epidermal Growth Factor) and 0.02 μg/ml bFGF (basic fibroblast growth factor) growth factors. The cells were seeded in 60 mm plates and cultured at 37° C. and 5% CO2.
The cells were kept in a humidified incubator for 48 h, and after this time they were recovered by centrifugation at 1000 rpm for 5 min, resuspended in neural induction medium (DMEM/F-12+GlutaMAX supplemented with 1% N2 (100×), 1% MEM-NEAA (MEM 100× non-essential amino acid solution) and 1 μg/ml heparin, seeded in 60 mm plates and kept in this culture medium for 48 hours at 37° C. and 5% CO2.
After this, the cells were cultured in Matrigel in 60 mm plates and in the presence of a differentiation culture medium. The composition of this medium was 50% DMEM/F-12+GlutaMAX and 50% neurobasal medium (1×), supplemented with 0.5% N2, 0.025% Insulin, 0.5% MEM-NEAA and 1% penicillin-streptomycin, 0.035% of 2-Mercaptoethanol (1:1000 dilution) in DMEM/F-12+GlutaMAX and 1% B27 without vitamin A. The neurospheres were kept in Matrigel for 72 h before being transferred to the bioreactor.
The neurospheres were kept in the bioreactor in the presence of a differentiation medium supplemented with B27 with vitamin A (Lancaster M A et al., 2014). The medium was changed every 72 h.
| Number | Date | Country | Kind |
|---|---|---|---|
| P201830866 | Sep 2018 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2019/070589 | 9/4/2019 | WO | 00 |
