Patent Application
20230416669
The subject of the invention is a cassette for growth and treatment of cells and a system for continuous, long-term cell growth and treatment, controlled by a computer application. The system is primarily used to confirm whether a given drug actually works in in vitro therapy, and to perform drug tests on cell culture and to prepare a drug concentration curve. The goal of the drug concentration curve is to mimic the in vivo ADME process of drug: absorption, digestion, metabolism, excretion. The device is to be used especially in the case of testing drugs used in the treatment of cancer cells or other disease models. Other large scale cell culture applications can be also explored like, for example, artificial meat cultivation.
Cancer is still the leading cause of death in the world. High morphological and phenotypic heterogeneity of the population of cancer cells, as well as their ability to develop genetic and epigenetic mutations increasing the prevalence of chemo-resistance are the main causes of failure of anticancer therapies. The occurrence of variability between the same types of cancer among patients makes it difficult to choose not only the right treatment regimens, but also to determine the effective dose of drugs. In addition, the available molecular biology methods that allow personalization of the treatment path for individual patients are not as effective as desired due to the time interval in generating data, which often leads to the development of secondary resistance to drugs and the formation of metastases. Given the above, the search for alternative methods that facilitate the selection of individual therapies is a sensible approach in the development of anticancer medicines. However, there remains one critical challenge that faces the oncology research and clinician communities in choosing the best chemotherapy regimen in addition to the complexities of cancer heterogeneity described above. This challenge concerns the disappointment when drugs shown to be effective against tumour cells in laboratory in vitro tests are often much less effective in the clinic. While part of this failure in drug efficacy is likely due to the differences in drug concentrations that tumour cells get exposed to in in vitro compared with in vivo scenarios, the larger cause of efficacy failure is very likely due to the substantial differences in cell and tissue organisation and diversity, gene expression and metabolic state found in patient tumours compared with the highly simplified 2D monolayer culturing commonly used in the drug discovery and characterisation process.
Examples of prior art solutions are known from documents such as EP3115449, WO2012033439, US20110217771 or EP1458483.
There is a growing need for 3D cell culture instead of 2D culture in order to begin to recreate the cell morphologies, behaviours, gene expression and metabolic profiles found in vivo.
There are different types of prior art receptacles or surfaces that have been developed for 3D cell culture including, for example: conventional petri dishes, conventional multi-well plates (6-well to 384-well), solid carrier beads or mats used inside walled containers, or modified glass or plastic microscope slides.
Also “Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device” M. Hagiwara et al. Appl. Sci. 2018, 8(2), 235 discloses 3D culture platform enabling large scale 3D imaging by fine spatial alignment of the image dataset obtained from multiple directions.
There are prior art designs that enable the supply of cell culture media and additional reagents to growing 3D cell cultures (and not just batch-wise replenishment of surface covering cell culture media), such as: synthetic hollow fibres (can be in layers or matrix layout) for supplying nutrients to microscope slide sized cultures; rolled wall vessels (creating micro zero gravity environment) containing 3D solid scaffold mat pieces supplied with pumped cell culture media; synthetic hollow fibres wrapped up ‘swiss-roll’ style with solid 3D scaffold mats inside a cylindrical vessel supplied with pumped cell culture media; sealed vessels containing microscope slide sized cultures through which cell culture media can be pumped and which provide for additional cell types to be grown in adjoining diffusion linked chambers; conventional continuous-flow stirred bioreactors that in principle can be used with cells grown on 3D scaffold coated beads, matting or grown encapsulated within protective gel beads, cell culture media pumped through extracellular matrix composed tubes contained within a vessel; supply and analysis of used cell culture media supplied by micro-diffusion channels constructed within microscope slide sized apparatus.
Examples of prior art supports and containers for cell cultures growth are known from: WO2015044813, EP3115449, WO2012033439, US20110217771, EP3019625, US20140141464, US20160139110 or EP1458483.
Prior art documents CN103077296 A and CN103077295 A concern a problem of simulating intravenous injection and oral administration pharmacokinetics model based on flow velocity regulation. These systems are based on pumping the fluids with one peristaltic pump and then mixing all components according to complicated dilution mathematical schemes. As a result the presented approach is very complicated.
Also document US2002/146817 discloses an automated bioculture and bioculture experiments system forming one, monolithic structure, wherein it is difficult to remove only the main components needed for example for starting new cell growth.
None of the known above technologies provide way of using 3D-matrices in hardware devices capable of supporting and monitoring 3D cell growth in a long term, researcher friendly manner. There is also no prior art giving at the same time a possibility to mimic real-life pharmacokinetics of a tested drug type. Most of prior art documents focus on microscale experiments, and as it is well known microscale does not always reliably reflects the phenomena occurring in macroscale.
The present invention addresses several aspects of the challenges listed above.
Firstly, in enabling the programming of clinically meaningful drug concentration dosing profiles and timings to cell cultures in a researcher friendly manner.
Secondly, the use of constant media flow allows the possibility of conducting long-term culture without the participation of a researcher. It can also allow the analysis of growth factors and metabolites secreted by tumour cells under the influence of various drug administration doses, and delivering additional relevant information to help determine the optimal anti-cancer treatment regimen.
Another goal of the invention is to provide an automated long-term 2D and 3D cell culture. Many cell types of importance to medical research require growth periods of several weeks or more. Whilst there are complex automated systems for non-3D cell growth (e.g. engineered stem cells), there are currently no programmable systems that cater for true 3D cell culture at a scale larger than microscope slides. It is also preferable that the system could fit conveniently in a standard laboratory humidified incubator.
Another goal of the invention is to provide an externally automated variable controlled flow and composition of two different cell culture media components. Although examples of pumped single component liquid addition to culture vessels exist already, there is no convenient way to pre-programme for example the pharmacokinetically relevant changing concentrations with time of therapeutic agent (or growth factor) in such a laboratory incubator sized device.
The technical problem solved by the current invention is: to recreate the in vivo pharmacokinetics using an in vitro device; to create very stable flows of cell culture media in a long periods of time like e.g. several months all run by the software; to provide an easy to use container (cassette) that once closed under sterile conditions can be then transported in a non-sterile environment and plugged into a pumping device without braking the sterility, wherein this cassette is more of a cell culture labware but not an entire device therefore it is much easier to handle, much lighter and permits to keep the main device with the computer means in the incubator all the time.
The invention is a cassette for growth and treatment of cells and ease of handling of cell culture outside the sterile cell culture cabinet and re-fitting to the device in the incubator. The cassette comprises at least one cuvette being a container for cell culture growth, a cell culture media reservoir, a reagent reservoir, a waste container, and interconnecting tubes enabling connection with peristaltic pumps. The cassette is detachable and movable as an entity. In the working state, the cassette is placed in a drawer, wherein the reagent reservoir is connected by tubing with a pump connected by tubing with the cuvette, and the cell culture media reservoir is also connected by tubing with another pump connected by tubing with the cuvette. Tubing can be easily detached from the pumps, which preferably are peristaltic pumps. After detaching the tubing from the pumps, the whole cassette can be easily removed from the drawer. The cassette may comprise a detachable holder attached to a side of the cassette suitable to connect the cuvette for cell culture. The shape of the holder will determine what kind of cuvette can be used.
The cuvette for cell culture growth of such cassette can be any suitable container or cell culture system known from the prior art which allows cell culture media to flow through the device and support the growth of cells. The only requirement is that the cuvette must have compatible input and output tubes connections or be provided with an adequate adapter.
Another aspect of the invention is the whole automated reagent delivery system for growth and treatment of cells, wherein the system comprises
Preferably, the two peristaltic pumps are
The constant fluid flow means that the total amount of fluid passing through the cuvette is constant. Preferably, the cell culture media flow is reduced accordingly when the reagent flow occurs by controlling the operation of the pumps. The tube conducting the fluid with cell culture media and the tube conducting the fluid with reagent are preferably merged into one tube before entering the cuvette to provide one, constant flow of the fluids within the cuvette. As a result of such setup no additional mixers are needed.
After passing through the cuvette, the fluid is directed toward the waste container. It is also possible to use a system of waste containers separating successive fractions.
Advantageous effects of the invention are
The medium is pumped very slowly (e.g. 1 ml/hour) into the cuvette. The cell culture media pump for pumping cell culture media into the cuvette is turned on at all times to keep the cells alive. The reagent (for example drug) pump for pumping a reagent into the cuvette does not need to be turned on all the time. Both pumps provide together a constant fluid flow, when the reagent pump starts working, the culture media pump slows down. Thus the total volume of fluid flowing through the cells is the same all the time.
Moreover, the system allows for the ongoing collection of data. The computer controller permits to easily compose the entire experiment by assembling it from parts (predefined blocks) with a resolution of preferably a few (e.g. 1 or 5) seconds for an experiment lasting even 72 hours. The software makes it possible to program a simulation of the entire (e.g. 3-week) therapy cycle without post-programming operation.
The invention is presented on figures of the drawing, wherein:
The system is designed to fit in conventional laboratory incubators as shown in
An exemplary cuvette (3) compatible with the invention may comprise a main container (5) and two end covers at inlet and outlet end of the main container (5), connected with the cell culture media input and output tubing. In the vertical configuration of the container we can call the end covers as a bottom end cover and a top end cover. The two end covers comprise seals that form a liquid and low-pressure resistant connection with the main container (5). Preferably the cuvette (3) comprises means for simple cuvette dis-assembly for retrieval and analysis of contents e.g. via easily removable cuvette's end covers (4).
The top end cover has at least one opening through which cells are injected, forming a top connection point (20) for the cell culture media supply or drain tube. There can be an optional connection (19) for a syringe filter placed at the top end cover—such connection (19) can be used only in the cuvette's versions where the cell culture media flows through the central vessel. Moreover, the same opening through which cells are injected can be used to accommodate a filter that has the purpose of buffering the pressure inside the cuvette—the opening can also be closed with a rubber stopper or a screw, especially when pumping the cell culture media through the hydrogel itself without a tubing, wherein maintaining pressure may be crucial. The central vessel can be connected and stabilized between the bottom and top end cover by mounting elements (25, 26) placed at the inner side of the covers. A bottom connection point (21), for the tube that supplies or drains the nutrient solution, is placed at the bottom end cover. At the top end cover there can be an opening (22) for adding a fill inside the cuvette, be it a hydrogel or injecting cells—this hole can also have a syringe filter mount so that a number of openings is reduced. At the bottom and top end there can be placed a net (23, 24).
The main container (5) of such exemplary cuvette is preferably optically transparent (for example is made of glass or plastic) for visual, microscopic, or metabolic imaging.
The central vessel is preferably made of proteins, and preferably transparent or translucent, could be also made from synthetic materials as long as they will allow for easy diffusion of the nutrients through the central vessel wall and supply of oxygen to the surrounding tissue.
Preferably, to provide a means of cell culture media being continuously supplied to and removed from cells growing within the exemplary cuvette (3) compatible with the invention on or in hydrogel (or other carrier), one or more of the following features are used to form at least one central vessel:
Such tube(s) (6) support growth of various cell types, facilitating (but not limited to) vascularisation, extravasation, intravasation, and tumour adherence.
It is also possible to provide hydrogel fragments that can simply permeate cell culture media through it instead of through fixed tubes (6). The means for macroscale 3D growth of cells may comprise hydrogel and pre-formed channels in the hydrogel containing inserts.
Flow of cell culture media can be directed via pre-formed channels in hydrogel containing inserts, and stacking of inserts within cuvette (3) facilitates i) increased surface area in contact with pumped oxygen and nutrient containing cell culture media ii) increased volume of hydrogel matrix interspersed with flowing nutrient iii) options for sandwiching of different hydrogel/different juxtaposed cell-type containing layers.
3D cell culture materials known from the prior art include use of animal derived extracellular matrix components (e.g. matrigel, collagen), plant derived 3D scaffolds (e.g. soft agar), synthetic extracellular matrix-like components (e.g. synthetic laminin, collagen, fibronectin), synthetic soft 3D cellular scaffolds (e.g. Peptide modified polyacrylamide gels), or synthetic hard 3D scaffolds (e.g. alvetex). The majority of the above materials can be well defined chemically and functionally, the exception being the variation seen from batch to batch of animal derived basement membrane preparations (variations in endogenous growth factor levels and possibly uncharacterised virus or antigen presence that may affect long term comparative experimental studies and in particular jeopardise animal studies). In general, 3D cell cultures according to the prior art are either grown by seeding on the surface of a matrix, by seeding within the body of a matrix, or by seeding in suspension cultures (whose volumes are often physically constrained).
Current preparation of 3D cell culture material for use in the desired experimental device may include: warming of extracellular matrix components to catalyse gel support formation, cooling of soft agars to permit gel formation, and chemical or photo cross-linking of scaffold components in situ or prior to dispensing scaffold. A protein hydrogel as described in documents EP3689971A1 and WO2020161613A1 can be used. The above procedures often allow pre-mixing of liquid components with cells prior to gel formation; the nature of the chemical or photo cross-linking determines whether or not cells can be added before gel formation. In the case of animal, plant and synthetic soft scaffold materials, each can normally be dispensed into the appropriate cell culture receptacle using conventional liquid handling techniques. In the case of synthetic hard cellular scaffolds these may be produced using techniques such as 3D-printing into culture receptacles or adding pieces of pre-formed scaffold as inserts into the culture receptacle.
The tubes with the cell culture media (supplying and discharging the cell culture media) can be attached to the cuvette (3) with the use of sleeves, wherein the inner diameter of the sleeve is slightly larger than the inner diameter of the tube supplying the nutrient solution.
Such exemplary cuvette (3) can be used for the cultivation of adherent cells or cells immobilized in a hydrogel or hydrogel fragments, wherein the cells can be human, animal, insect, or plant cells, or their combinations—additionally, bacterial cells can be added, especially when cell culture creates a research model, e.g. bacterial infection.
The exemplary cuvette (3) can be attached to the cassette (2) via main and bottom grips of the cassette (2) that fit closely to the tube mounting sleeves—such mounts are centrally located in relation to the axis of rotation of the cuvette, thanks to which its imaging will be more precise.
There is also a possibility to use any other suitable container or cell culture system known from the prior art which allows for cell culture media to flow through the device and support the growth of cells, instead of the exemplary cuvettes (3) presented on
The cassette (2) preferably has a positioning system, for example guides and magnets on the sides that position it within the drawer. Precisely repeatable positioning of the cassette (2) within the drawer is important for the imaging system performance.
The cell culture media reservoir (9), the reagent reservoir (10), and the waste container (8) preferably comprise filters (e.g. 0.2 μm) preferably placed in their covers. Filters provide sterility inside the bottle, but also give the possibility of stabilizing the pressure in the bottle when the cell culture media is pumped into it and pumped out of the reservoirs and containers.
The cassette's (2) design facilitates sterile work. It allows for preparation of the test system (opening and closing of cuvette, reservoirs) under sterile conditions (in the air flow cabinet), and transporting it (in non-sterile conditions) to the device containing pumps. All elements are compactly arranged in one place, which facilitates the ergonomic handling of the entire cassette (2). It is also possible to connect the cassette (2) to the pumps (13, 14) in a non-sterile incubator while maintaining the sterility of the entire internal culture system.
The automated reagent delivery system for growth and treatment of cells according to the invention comprises at least one slide out drawer (1), at least two peristaltic pumps (13, 14) attached to the drawer (1), and the cassette (2) attached to the drawer (1) and connected with the peristaltic pumps (13, 14), a computer controller and computer program product (15). The computer program product comprises means for manual and automatic control of liquid flow rates, output composition between the independent liquid reservoirs, pump control and execution of pre-set liquid flow programs, customization and saving of new automated liquid handling programs. There is a possibility of digitally saving complete programmed segments of the pumping protocol, e.g. in 24-hour segments. There is also a possibility of recording all flow data—the electronics are coupled in such a way that it allows one to plan everything from the very beginning to the very end. It is a huge advantage over other systems that, for example, require human intervention in order to complete a long-term experiment with the risk of errors.
While the system is in use, first of all, a program is created and the experiment plan is electronically recorded. It can also simply be copied and used or, in addition, it can be edited and modified for future experiments. The software is created as “blocks”—elements of the experiment that can be freely copied/modified/saved. These features provide a rapid way to create, modify and execute experiments without error.
In the event of a power failure the system will restart in the same place after power resumes, and the information about the power outage will be included in the final experiment report automatically generated by the program.
The pumps used in the system can be a cell culture media pump (13) for pumping cell culture media into the cuvette (3), and a reagent pump (14) for pumping a reagent into the cuvette (3). The cell culture media reservoir (9) is thus connected with the cuvette (3) via a culture media input tubing and a cell culture media pump (13), and the reagent reservoir (10) is connected with the cuvette (3) via a reagent tubing and a reagent pump (14). Tubing (11), meaning the culture media input tubing and the reagent tubing, may be closed in tubing housing units (12) that simply unclip/clip onto pump heads. Preferably, both pumps (13, 14) provide together a constant fluid flow. The mixing of the reagent fluid with cell culture media occurs in the overlapping segment of the input tubes before entering the cuvette (3).
The performance of the system in visible on graphs on
| Number | Date | Country | Kind |
|---|---|---|---|
| 20461583.5 | Nov 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/060754 | 11/19/2021 | WO |
