CASSETTE AND SYSTEM FOR GROWTH AND TREATMENT OF CELLS

Abstract

A cassette for growth and treatment of cells having at least one container for cell culture growth, a cell culture media reservoir, a reagent reservoir, a waste container, and interconnecting tubes enabling connection with peristaltic pumps, wherein the cassette is detachable and movable as an entity. Also disclosed is an automated reagent delivery system for growth and treatment of cells, wherein the system includes at least one slide out drawer, at least two peristaltic pumps attached to the drawer, the cassette according to the invention attached to the drawer and connected with the peristaltic pumps, a computer controller and computer program product having 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.

Claims

1.-7. (canceled)

8. An automated reagent delivery system for growth and treatment of cells, wherein the system comprises: at least one slide out drawer, at least two peristaltic pumps attached to the drawer, the cassette for growth and treatment of cells comprising at least one cuvette for cell culture growth, a cell culture media reservoir, a reagent reservoir, a waste container, and interconnecting tubes enabling connection with peristaltic pumps, wherein the cassette is detachable and movable as an entity, wherein the cassette is attached to the drawer and connected with the peristaltic pumps, and a computer controller and computer program product comprising 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.

9. The system according to claim 8, wherein the two peristaltic pumps are a cell culture media pump for pumping cell culture media into the cuvette, and a reagent pump for pumping a reagent into the cuvette, wherein the cell culture media reservoir is connected with the cuvette via a culture media input tubing and a cell culture media pump, and the reagent reservoir is connected with the cuvette via a reagent tubing and a reagent pump, and both pumps provide together constant or pulsating fluid flow.

10. Use of the system according to claim 8, for mimicking pharmacokinetics or in vivo ADME processing of a drug.

11. Use of the system according to claim 8, for large scale cell manufacturing, microorgans growth of embryonic cells.

12. Use of the system according to claim 8, for cells growth in suspension.

Description

[0036] The invention is presented on figures of the drawing, wherein:

[0037] FIG. 1a view of the system fitted into a conventional laboratory incubator;

[0038] FIG. 2a view of the system with one of three drawer units pulled out;

[0039] FIG. 3a view of the unit comprising a slidable drawer with a removable cassette;

[0040] FIG. 4a view of the removable cassette;

[0041] FIG. 5an exploded view of an exemplary cuvette compatible with the invention;

[0042] FIG. 6a view of the exemplary cuvette's inlet end cover and outlet end cover;

[0043] FIG. 7an exploded view of another exemplary cuvette compatible with the invention;

[0044] FIG. 8an exploded view of another exemplary cuvette compatible with the invention.

[0045] FIG. 9Aa graph presenting mean plasma concentrations (+SE) on day 1 after oral administration of imatinib at doses of 400 mg and 500 mg [DOI: 10.1200/JCO.2004.03.050 Journal of Clinical Oncology 22, no. 5 (Mar. 1, 2004) 935-942].

[0046] FIG. 9Ba graph presenting a replication of observed plasma concentrations using system according to the invention.

[0047] The system is designed to fit in conventional laboratory incubators as shown in FIG. 1. The system comprises at least one and preferably three or six units in a form of slide out drawers, as shown in FIG. 2. Each unit comprises a removable cassette (2) designed so that only one essential component for aseptic handling needs to be transported into an air-flow cabinet for aseptic liquid replacement, additions and disposals, cell or tissue injections and cuvette (3) removal for imaging and further manipulation. Each removable cassette (2) comprises a cell culture media reservoir (9), a reagent reservoir (10), at least one cuvette (3) and preferably a waste container (8), accordingly interconnected by tubing. The cell culture media reservoir (9) is connected with the cuvette (3) via a culture media input tubing and a cell culture media pump (13) for pumping cell culture media into the cuvette, and the reagent reservoir (10) is connected with the cuvette (3) via a reagent tubing and a reagent pump (14) for pumping a reagent into the cuvette (3). Pumped fluids after passing through the cuvette (3) can be directed via output tubing into the waste container (8) or again into the cell culture media reservoir (9). Preferably, removable cassette (2) comprises a grab point (18) enabling easy handling. The grab point (18) can be in a form of one or more handles or openings enabling easy grabbing and carrying of the cassette (2), or any other suitable means placed located in any suitable place on the outer surface of the cassette (2).

[0048] 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).

[0049] 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 coversuch 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 cuvettethe 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 cellsthis 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).

[0050] 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.

[0051] 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.

[0052] 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: [0053] extracellular matrix component tube(s) (6) ranging from 1 mm to 10 mm diameter to carry cell culture media in and out of cuvette (3), at flow rates that maintain high concentration gradients for diffusion of components to and from growing cells; [0054] choice of single tube or multiple tubes, with or without opening/shutting valve ability; [0055] plastic fabric tubing, dialysis tubing.

[0056] Such tube(s) (6) support growth of various cell types, facilitating (but not limited to) vascularisation, extravasation, intravasation, and tumour adherence.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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 combinationsadditionally, bacterial cells can be added, especially when cell culture creates a research model, e.g. bacterial infection.

[0063] 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 sleevessuch mounts are centrally located in relation to the axis of rotation of the cuvette, thanks to which its imaging will be more precise.

[0064] 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 FIGS. 5-8.

[0065] 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.

[0066] The cell culture media reservoir (9), the reagent reservoir (10), and the waste container (8) preferably comprise filters (e.g. 0.2 m) 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.

[0067] 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.

[0068] 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 datathe 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.

[0069] 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 blockselements of the experiment that can be freely copied/modified/saved. These features provide a rapid way to create, modify and execute experiments without error.

[0070] 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.

[0071] 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).

[0072] The performance of the system in visible on graphs on FIGS. 9A and 9B. The graph of FIG. 9A presents mean plasma concentrations (+SE) on day 1 after oral administration of imatinib at doses of 400 mg and 500 mg bid. The x-axis shows the time in hours (h). The curve marked with circles () presents results for 400 mg dose and the curve marked with squares () presents results for 500 mg dose. The source of the graph is DOI: 10.1200/JCO.2004.03.050 Journal of Clinical Oncology 22, no. 5 (Mar. 1, 2004) 935-942. The observed plasma concentrations were replicated using the system according to the invention. Results of mimicked pharmacokinetics are presented in FIG. 9B.