Patent Application
20130203106
This invention relates to experiments and tests with live cells, including mammalian derived cells and in particular it provides the method and apparatus for delivery of liquids such as cell culture liquids and other reagent containing liquids. The experiments and tests can include biological experiments, experiments for medical research, drug development, diagnostic tests and other such similar experiments. The invention can also be used for cell production, tissue culture and protein drug production. The invention can be used with manual-controlled or programmed pumps to feed cells placed in conventional laboratory tissue culture flasks, Petri dishes and multi-Petri dishes plates. The invention can also be used with WIFI technologies for control of the pump. The system can be applied in the field of bioreactors, where it is necessary to separate incoming fresh medium and the outgoing media containing by-products of biological cells growth.
In modern cell biology, biotech, drug development or medical laboratory small-scale cell culture is performed using a variety of specialized containers such as cell culture flasks, Petri dishes or multiwell plates. These containers come in different volume sizes and with different surface treatments optimized for specific types of cells and particular experiments. Usually a cap or a lid of the tissue culture container is gas permeable or it is not air-tight sealed against the container's body. This ensures exchange of air between the interior of the container and the ambient when the container is placed inside culture incubator. Such exchange of air is important for supporting appropriate conditions for cell life and growth, maintaining the desired pH in the culture medium release of gas originating from the biological processes within the media. Furthermore, cell incubators are often equipped with gas controllers for mixing of nitrogen, oxygen and carbon dioxide gases at predetermined ratio to support cell culture.
In addition to delivering gas to the growing cells, the culture media contains nutrients and salts necessary for the cell growth and division. It may also contain other liquids necessary for biological experiments. As the cells undergo their life cycle, the medium becomes starved of nutrients and also filled with cell by-products, which need to be disposed. The cell growth rate is largely dependent on the cell type. Some cells are slow growing and their doubling time is high: Hep-G2 Liver cancer (48 hrs), MIA PaCa-2 Epithelial carcinoma (48 hrs), Mesenchymal Stem cells (45-68 hrs). Some cells are moderate growing: MCF-7 Breast cancer (29 hrs), LNCaP Epithelial carcinoma (35 hrs), MDA-MB-231 (30 hrs) ; and fast growing: Human Embryonic stem cells (12-18 hrs).
Depending on the cell type in the culture, the medium inside each flask (cell container) may need to be exchanged for a fresh culture medium typically every 10-14 hours. This routine of laboratory cell culture is carried out inside a tissue culture laminar flow hood to ensure aseptic conditions and it is often manual and laborious.
Some cell types require continuous supply of fresh medium, and disposal of a used medium to ensure their physiological growth. Such cells types are liver cell (hepatocytes) and embryonic stem cells. The growth of these cells is difficult to sustain when manual culture techniques are applied. Successful cultivation of different cell types demands not only skillful handling, but also depends to a large extent on the chosen cultivation system. Continuous perfusion technology closely mimics the physiological conditions within the body enabling production of drastically increased cell densities and more organotypic cell morphologies, sometimes even to the extent of achieving 3-dimensional cell growth.
Recent advances related to automation of cell culture can be divided into two distinct groups.
First group of inventions is focused on improvements to tissue culture vessels to aid automatic withdrawal of culture medium.
U.S. Pat. No. 6,908,767 B2 to Bader et al (2005) describes method for growing cells in an automated manner for diagnostic purposes. A plate having plurality of holes or wells is used to culture cells. Unlike commonly used multiwell plate, the bottom of the plate described in the invention, is gas permeable to allow transfer of oxygen to the cultured cells. Additionally, the individual wells need to be sealed in order to inject and withdraw the culture medium. The invention describes ways by which the well can be sealed and also method to ensure that the culture medium delivers nutrients to the cells uniformly. The drawback is that the exact amount and proportion of carbon dioxide and oxygen delivered to cells is unknown. It depends on several parameters such as permeability of the film, flow rate of delivery of gas and also on amount of medium and the rate of flow of medium in the well. Control of oxygen to carbon dioxide ratio can be important in order to support sustain desired PH of the medium, one of the key parameters for cell culture. Additionally the patent describes the use of membrane bottom of a well to deliver the gas to the cells. It is unclear how the well can be sealed, as pressure during perfusion can eventually transfer the medium outside the well through the porous membrane.
U.S. Pat. No. 7,749,750 B2 to Kobayashi et al (2010) describes a culturing apparatus having two distinct parts: a culture vessel having the elastic seal bonded to it with two ports for injection and withdrawal of culture medium and the second part having microinjection means capable of connecting to the delivery system. The two parts are sealed via the elastic seals, which provide a convenient method for robotic automation, which is also described in the invention. Additionally the system introduces seals allowing access to the culture vessel by robotic dispensers and at the same time ensuring sterile operation. It is unclear form the invention how the necessary conditions of the cell culture are supported: temperature, PH and gas constitution of the culture medium.
US patent 2007/0031963 A1 to Chang et al. (2007) describes cell culture flask modified for various automated processes such as introduction and removal of culture medium. The access to the cell culture vessel body is allowed via specially designed closures, which can accommodate blunt tips and close after withdrawal of the tip from the cell culture vessel body. The closures are pre-pierced to allow easy access with blunt pipette tips. These advanced cell culture vessels are used without the lid, which eliminate the need for using robotic arms to automating cell culture.
U.S. Pat. No. 7,816,128 B2 to Nakashima et al. (2010) does not deal with modifications to cell culture vessels, but provides method of automated cell culture using robotic apparatus capable of conveying culture vessels. The system consists of at least one incubator capable of accommodation culture vessel, the dispensing and suction machine and robot for opening lids of culture containers such as Petri dishes.
Second group of inventions is focused on the design of integrated systems containing purpose built cartridge, bioreactors or perfusion chambers to host the cells. These systems are usually fully enclosed and include perfusion pump, system of valves and gas control apparatus.
U.S. Pat. No. 7,270,996 B2 to Cannon et al (2007) provides the bio-culture platform including closed-loop flowpath cartridges for growing cells, system for sampling and collection of culture medium capable of integration with analysis system for evaluating the status of growing cells. The cartridges used for growing cells are connected via series of ports to a perfusion path capable of circulating the culture medium and also maintaining required oxygen and carbon dioxide concentration via oxygenator units. The medium can be delivered through the closed-loop flowpath once or repetitively. The medium reservoir is provided as the source of culture medium connected via oxygenator to the bioreactor cartridge output of which is connected to the waste reservoir. There are embodiments with valves introduced into the flow path to divert culture medium for PH or other analysis sampling. Although, the invention provides an integrated culture system, it requires purpose built cartridges and multiplicity of auxiliary components to operate. Additionally an oxygenator module would only operate successfully if medium flows through it continuously; otherwise there will be a difference in medium oxygenation inside the oxygenator and in the cartridge where cells grow. This may lead to consumption of large amount of fresh medium if the system is used in the single pass flow mode.
Another integrated bio-culture system is described in US patent 2011/0212493A1 to Hirschel et. al. (2011). The invention includes construction of bioreactor integrated with the cell culture system. Similar to the previously described patent the flow path includes auxiliary components to ensure the correct pH and oxygen concentration of medium. A particular feature of this patent is that the design of the bioreactor perfusion chamber allows for growth of significant amount of cells due to the large surface area provided for the growth. This is achieved by spiraling the interior surface of the bioreactor and also by pleating the said surface.
Despite all the efforts in developing automated cell culture systems, majority of routine cell culture is still done manually. In practice, the main obstacle in implementing automated cell culture system is cost associated either with equipment or consumables. Recent technological advances in this area usually do not require costly or elaborate flasks or cell culture vessels but they do require complicated robotic system to automate operation of the cell culture apparatus and enable replacement of the culture medium. In the case of integrated systems and bioreactors, the cell culture containers are purpose built, often limited to culture of a particular cell type and are costly.
The present invention provides a novel system and method for laboratory-scale cell culture compatible with conventional tissue culture containers (flasks) available on the market and routinely used in cell biology laboratories or in medical laboratories. The system and method for laboratory-scale cell culture is further compatible with conventional cell culture incubators and can be used for a variety of adherent and non-adherent cell types. The invention further typically provides a system with a low dead volume and capable of handling low volumes of cell culture media, comparable to the ones used in static cell culture experiments.
The present invention further typically provides system and method for laboratory-scale automated cell culture without use of robotic arms, dispensers or other complicated robotic machinery. This minimizes the cost of cell culture instrumentation and routine and allows the system to be applied in small- and medium scale laboratories.
All the media-carrying components of the system, excluding the pump, such as tubing, bottle and cap components can generally be disinfected by autoclaving and therefore can be reused. The pump can be disinfected by washing with appropriate disinfectant fluid and cell culture flask, which is disposable.
The system allows both for introduction of fresh culture medium and removal of the culture medium containing by-products of cell life-cycle using a single pump, without use of valves.
The system allows continuous perfusion of fresh culture medium and withdrawal of the culture medium containing by-products of cell life-cycle, which allows growing cell under more physiological conditions.
The invention provides cell culture system for laboratory-scale bioreactor applications, where outgoing consumed culture medium does not come in contact with the incoming fresh culture medium and outgoing media contains proteins, enzymes, hormones and antibodies that need to be collected for disposal or for further analysis.
The system allows for remote programming of the perfusion time and number of perfusion cycles per day using Smartphone connected to the pump controller via Wi-Fi network.
Broadly, the invention provides an apparatus suitable for performing experiments on live cells comprising:
a cell culture vessel;
a fresh culture medium storage vessel;
a used culture medium collection vessel;
a supply conduit adapted to provide fluid communication between the fresh culture medium storage vessel and the cell culture vessel, an inlet of the supply conduit ideally being located towards a base of the fresh culture medium storage vessel;
a drainage conduit adapted to provide fluid communication between the cell culture vessel and the used culture medium collection vessel, an inlet of the drainage conduit being disposed within the cell culture vessel such that in use it is it located in the culture medium; and
pump means adapted to pump fresh culture medium from the fresh culture medium storage vessel to the cell culture vessel and pump used culture medium from the cell culture vessel to the used culture medium collection vessel.
Preferably, the cell culture vessel is not sealed to ambient (i.e it is not airtight), and the cell culture storage and collection vessels are generally pressurized during use. This is generally achieved by providing a pressure equalization conduit between the storage and collection vessels such that the pressure exerted on one of the vessels by the pump is transferred to the other vessel. In this embodiment, the storage and collection vessels need to be sealed to the outside. Generally, the ends of the pressure equalization conduit are located in the headspace of the respective vessels, although in one embodiment one or both of the ends may be located within culture medium.
However, the apparatus may also be operated in a manner in which the cell culture vessel is sealed to ambient (i.e. it is airtight). In this case, during operation the pump will pressurize the cell culture vessel, and the pressure will force culture medium out of the vessel via the drainage conduit. In this embodiment, the storage and collection vessels may be connected by a pressure equalization conduit, or they may be open to ambient (i.e. not pressurized).
In one embodiment, the fresh culture medium storage vessel and used culture medium collection vessel are formed by separate compartments of the one container.
The invention also relates to a method of perfusing cells in a cell culture vessel with fresh cell culture medium, which method employs an apparatus according to the invention and comprises the steps of:
adding fresh culture medium to the fresh culture medium storage vessel such that the inlet of the supply conduit is immersed within the culture medium;
adding fresh culture medium to the cell culture vessel such that the inlet of the drainage conduit is immersed within the culture medium; and
actuating the pump to pump fresh culture medium from the storage vessel to one end of the cell culture vessel and withdraw used cell culture medium from the second end of the cell culture vessel.
The cell culture vessel is typically a cell culture flask or a Petri dish, although the use of other cell culture vessels is envisaged.
In one embodiment, the pump is provided in-line in the supply or drainage conduit, ideally the supply conduit.
The pump is typically a solenoid pump or a peristaltic pump, although the use of other types of pumps is envisaged.. The pump is adapted to be actuated periodically, for example once every 5 mins, 10 mins, 30 mins, hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours. The pump may also be adapted to be remotely operated by means wireless communications, for example over a WI-FI network.
Suitably, the fresh culture medium is a liquid suitable for culturing live cells.
In another embodiment, the fresh culture medium contains a drug substance and the apparatus is used for monitoring cell status over time.
In another embodiment, the fresh culture medium contains substances required for promoting cell growth and division.
In another embodiment, the fresh culture medium contains a substance capable of inducing cell differentiation between different cell types.
In another embodiment, the fresh culture medium also contains a substance capable of inducing cells to produce cytokines, proteins, antibodies or other cell life cycle by-products.
The invention also provides a method of performing a toxicity assay on live cells, which method employs a method of perfusing live cells with cell culture medium according to the invention, wherein the cell culture medium includes a test agent, and wherein the method includes a step of assaying the toxicity of the test agent to the cells in the cell culture vessel.
Typically, the test agent is selected from a toxin or a drug, although other test agents may be employed for example food additives, agents found in industrial effluents, bacterial or viral components, and the like.
Also provided is an apparatus for culture of live cells comprising a pump, a culture medium vessel, a culture medium collection vessel, a cell culture vessel and conduits whereby:
Typically, the conduits do not link into the cell culture vessel in air tight manner
Suitably, the pump is installed in one of the conduits transferring the cell medium.
Generally, the conduit connecting the culture medium vessel and the culture medium collection vessel is not filled with culture medium during the operation.
The cell culture vessel is typically a cell culture flask or a Petri dish.
The pump is selected from a solenoid pump or a peristaltic pump.
The culture medium vessel and the culture medium collection vessel suitably form one vessel with one compartment forming culture medium vessel and the other one the culture medium collection vessel. Ideally, the conduit connecting the culture medium vessel and the culture medium collection vessel represents an opening in the partition separating the two vessels.
Generally, the outlet of the conduit connecting the culture medium vessel and the cell culture vessel is not placed in direct contact with the culture medium in the cell culture vessel.
Ideally, pump controller is capable of being programmed over the WIFI network.
Also provided is a method for sustaining cell life or cell culture work utilizing connecting a cell culture vessel by one conduit to a culture medium vessel and by a second conduit to a culture medium collection vessel, and connecting the culture medium collection vessel by a conduit to the culture medium vessel, and connecting the culture medium vessel by a conduit to the culture medium collection vessel and installing a pump in one of the conduits capable of transferring a volume of cell culture from the culture medium vessel to the cell culture vessel and then to the culture medium collection vessel.
Suitably, the conduits are connected to the culture medium vessel in air tight manner
Generally, the conduits are connected to the culture medium collection vessel in air tight manner.
Typically, the cell culture vessel is not sealed from the ambient in air tight manner.
Ideally, the pump is activated from time to time over the WIFI network.
Typically, the pump delivers flow from the culture medium vessel to the culture medium containing vessel at pre-determined intervals of time.
Suitably, the opening of the conduit connecting the cell culture vessel with the culture medium collecting vessel is coupled into the cell culture in the cell culture vessel.
References cited:
The invention will be more clearly understood from the following description of some embodiments thereof given by way of example only with reference to the accompanying figures in which:
a Image of MDA-MD-231 cell line in the T75 cell culture flask 2 hours after seeding
b Image of MDA-MD-231 cell line in the T75 cell culture flask after 96 hours of perfusion
a Image of WM793 cell line in the T75 cell culture flask 2 hours after seeding
b Image of WM793 cell line in the T75 cell culture flask after 130 hours of perfusion
The invention can be best understood from the description of the following drawings showing a number of embodiments. The embodiments given do not form an exhaustive list but rather are examples.
The term “culture medium” as used throughout this document should be understood in a broad sense as being any liquid, generally an biological liquid, including liquids for supporting cell growth or liquid for cell assays or liquid containing drugs. Likewise, the term “performing experiments” should be understood to include culturing cells, performing experiments and tests on cells, and observing the effects of various effectors on cell growth. Thus, for example, the apparatus is suitable for performing toxicity studies on live cells where the culture medium being provided to the cells includes a test agent, for example a putative toxin or a drug.
The term “monitoring cell status” should be understood to mean monitoring cell growth, cell death, apoptosis, cell activation, cell morphology, cell motility, and the general or specific production by the cell of molecules such as proteins, sugars, hormones, antibodies, and the like.
The term “cells” may be eukaryotic or prokaryotic cells. Generally, they are mammalian cells, usually human cells, although the apparatus and methods of the invention may be employed for performing experiments on other types of cells, for example bacterial or viral cells.
It is not necessary to seal the distal end of the pump outlet conduit 10 against the inlet port 8a of the cell culture vessel in airtight manner and likewise the culture medium collection conduit 12 does not need to be sealed in air tight manner against flask outlet port 8b of the cell culture vessel.
Before commencing the cell culture experiment, the fresh culture medium storage vessel 3, the used culture medium collection vessel 4, cell culture vessel cap 8, bottle cap 6 and bottle cap 7 and all the tubing are autoclaved to ensure sterility. Alternatively, these can be taken from sterile packs if disposable such components are being used. The pump 2 is washed with the disinfectant solution, for example 70% ethanol, and then flushed with air to remove excess disinfectant. The culture medium storage vessel 3 is filled with fresh culture medium 3a preferably with the neck of the vessel being left empty. The bottle cap 7 is applied to the fresh culture medium vessel 3 in air tight manner. The conduits 11 and 9 are connected according to the description above so that the opening 9a of the pump inlet conduit 9 is immersed into the fresh culture medium 3a and the opening 11 a of the pressure transfer conduit 11 is not. The culture medium collection vessel 4 is empty or near to empty. The cell culture vessel 5 is filled with live cells 5b and the cells are allowed to settle and attach to the bottom of the cell culture vessel 5. It will be appreciated by those skilled in the art that the cells may need to be placed on scaffold or matrix and reagents may need to be added to stimulate the cell experiments, or cell growth or cell assay. Details of the specific arrangements depend on the type of assay to be carried out.
During the experiment, the pump 2 starts operating and withdraws a preset volume of fresh culture medium from the culture medium vessel 3 and transfers it to the cell culture vessel 5. The pump outlet conduit 10 delivers fresh culture medium to the cells. The pump outlet conduit 10 can be in contact with cell culture medium 5a or be placed out of direct contact, e.g. above surface of the cell culture medium 5a. The latter case can be advantageous for bioreactor applications. As the pump 2 withdraws the volume of medium from the fresh culture medium vessel 3 it creates reduction in pressure of the gas directly above the fresh medium culture medium 3a. This reduction in pressure is transferred to the used culture medium collection vessel 4 via pressure transfer conduit 11. As culture medium collection conduit 12 is immersed in the cell culture medium 5a, the reduction in pressure inside the culture medium collection vessel 4 results in the flow of medium from the cell culture vessel 5 to the used culture medium collection vessel 4. The direction of the flow is indicated by arrows in
The used culture medium 4a has no direct contact with cell culture medium 5a. This allows the apparatus 1 to ensure that the waste products or by-products of cell life cycle are not affecting cell growth.
The system described above is capable of maintaining culture of various cell types. Following are two example of adherent cell culture using MDA-MD-231 breast cancer cell line and WM793 skin cancer cell line. Prior to the experiment MDA-MD-231 cells were first grown in growth medium (RPMI 1640, FBS-10%, 2 mM L-glutamine, 100 μg/ml Penicillin/Streptomycin). The cells were trypsinized using Trypsin-EDTA (0.05%) solution and resuspended in 20 ml of medium in T-75 cm2 flask. Flask was transfered to CO2 incubator and incubated for two hours for the cells to attach to the surface.
Similar technique was used for culture WM793 skin cancer cell line. Prior to the experiment cells were first grown in growth media (RPMI 1640, FBS-10%, 2 mM L-glutamine, 100μg/ml Penicillin/Streptomycin). The cells were trypsinized using Trypsin-EDTA (0.05%) solution and resuspended in 20 ml of media in T-75 cm2 flask. Flask was transfered to CO2 incubator and incubated for two hours for the cells to attach to the surface.
The described system can also be used for production of antibodies, cytokines, enzymes and hormones. Below, we give two examples of such applications for monoclonal antibody and virus production:
Monoclonal antibodies are antibodies with a defined specificity derived from cloned cells or organisms. They can be obtained from immortalised B-lymphocytes that are cloned and expanded as continuous cell lines (murine and human monoclonal antibodies) or from rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies). The monoclonal antibodies are produced by Hybridoma cellines, which have to be cultured for several days to get the maximum and optimal production of desired antibody. A wide range of anti-mouse and anti-human monoclonal antibody producing hybridoma cell lines are available. For example Hybridoma celline 60H9(9)D10.E6 (Derived from a patient with chronic Hepatitis C) is cultured using media RPMI 1640+2 mM Glutamine+1% Non Essential Amino Acids (NEAA)+1% Sodium Pyruvate (NaP)+20 u/ml IL-6+10% Foetal Bovine Serum (FBS). The product obtained after continuous culture for 10-15 days is Immunoglobulin G (IgGl) (kappa), which is specific for Hepatitis C virus NS4 region. Numerous research applications, including small-scale virus production, rely on T-flasks for cell culture. One example is the production of Adenovirus. HEK293 cells are grown to 80% confluence in T75 culture flasks. GFP adenovirus is added at 100:1 multiplicity of infection and the cells have to be perfused for 3 to 4 days for optimal production of viruses. Similarly Vero cells (African GreenMonkey, adult kidney, epithelial) are used to produce polioviruses.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12153868.0 | Feb 2012 | EP | regional |
