Cell retention device

Abstract

The present invention relates to a cell retention device for use within a culture vessel of a microbioreactor, to a microreactor comprising said cell retention device, and to a system comprising a plurality of microreactors each comprising said cell retention device. The present invention also relates to the use of the cell retention device of the invention for filtering culture medium out of a microbioreactor and to the use of the microbioreactor comprising the cell retention device of the invention for seed train culture, inoculum train culture or a main fermentation (production phase). The present invention further relates to a method of exchanging culture medium in a microbioreactor comprising the cell retention device of the invention.

Description

[0001] CELL RETENTION DEVICE

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of cell culture, and more particularly to a cell retention device for use within a culture vessel of a microbioreactor. The invention also relates to the use of the cell retention device and to methods using the cell retention device.

[0004] BACKGROUND OF THE INVENTON

[0005] Cell culture technique is an established method for biopharmaceutical manufacturing and is routinely used for producing recombinant proteins like e.g. receptors, enzymes, coagulation factors or antibodies used in research, as diagnostic tools or as therapy, as well as vaccines. Since then, the techniques have been continually improved to increase productivity and to decrease cost. Chinese hamster ovary cells (CHOs) have broadly been used as an expression host to make many of these products. For many years, the most common production process was fed-batch culture using suspension cells. Traditional fed-batch bioreactors consist of tanks that are usually between 2,000- 25,000 liters. Production phase typically lasts between 7 and 21 days by which time nutrients have been consumed and toxic metabolites have accumulated. During the production run, cells secrete the protein of interest into the cell culture medium and at the end of the run the secreted protein is separated from cell mass and cell culture fluid and further purified. Typical product titers are in the range of 1 to 5 grams per liter depending on the clone and antibody. While regular improvements have moved product titers from under 1 gram per liter to where they are now, increasing product titers even further would lead to reduction of manufacturing costs and would improve sustainability of recombinant protein production. . While improvements have continued in areas including advancements in cloning, cell line development, media formulation, removal of animal components, and downstream purification resins and columns, there has been little change to the actual fed-batch paradigm, with the exception of the emergence of perfusion reactors.

[0006] In contrast to traditional fed-batch systems, perfusion bioreactors enable cultivation of cells over much longer periods, even months, by continuously feeding the cells with fresh media and removing spent media while keeping cells in culture. Perfusion is therefore a continuous culturing method in which cells are either retained in the bioreactor or fed back into it. The harvested medium (called “permeate”) thus contains no cells, resulting in higher cell concentrations in a bioreactor while keeping the bioreactor volume low. Because of these higher cell concentrations such processes are characterized by higher productivity compared to fed-batch processes, expressed in grams product per liter culture volume per cultivation day. There are different ways of keeping the cells in a bioreactor while removing spent media, so in other words, different ways to execute a perfusion cell culture process. One way is to keep the cells in the bioreactor by using capillary fibers or membranes, which the cells bind to. Another way does not involve binding the cells, but rather relies on filtration systems that keep the cells in the bioreactor while allowing the media to be removed. Another method is the use of a centrifuge to separate cells and return them to the bioreactor. In general, filtration is done for clarification, selective removal and concentration of certain constituents from the culture media or to modify the media prior to further processing. In perfusion bioreactors however, filtration may also be used to enhance productivity by maintaining a culture in perfusion at high cell concentration. A wide variety of filtration systems exist that are adapted to large-scale filtration of media across various applications. However, conventional systems generally are ill-suited to filtering minute amounts of media in microscale cultivation systems. In addition, to accomplish concentration and / or sampling of small amounts of media, or perfusion of cells in cell culture media, conventional systems often have complicated structures that are difficult to manufacture and maintain, or are ill-suited for application to small volumes (e.g., microbioreactors holding 15 mL of fluid or less). A conventional workaround for these situations is the use of laboratory centrifuges to process such small culture volumes. The cells are pelleted in the centrifuge to allow the exchange of the spent culture medium with fresh medium. However, this approach is cumbersome, the medium exchange is not 100% complete and it is not a continuous process. Because of that, it has to be repeated frequently to simulate a continuous perfusion and the shear forces during centrifugation can be stressful to the cells.

[0007] Another approach is to use tangential flow filtration (TFF). Tangential flow filtration (TFF), also known as Cross-flow filtration, is a process of separation widely used in biopharmaceutical and food industries. It is different from other filtration systems in that the fluid is passed parallel to the filter, rather than being pushed through a membrane perpendicularly, as in the dead-end filtration, which is more prone to filter fouling. TFF is preferred also for its continuous filtration and reproducible performance. The particles that pass through the membrane, the permeate, are put off to the side, while the rest, the retentate, is recycled back to the feed. One example of TFF is alternating tangential flow filtration (ATF).

[0008] ATF uses a similar technique as TFF. The primary difference is that ATF is a filtration process, where the flow over the membrane is periodically reversed, which minimizes filter fouling. In ATF, the permeate is removed to a collection vessel while a diaphragm pump alternates between positive pressure and vacuum to move the retentate back and forth across the membrane. The retentate returns to the bioreactor between each cycle, mixing with the next fluid batch to be filtered. ATF is suited for collecting the desired product within the permeate, as the retentate is continually pushed back into the bioreactor. The alternating flow of the retentate cleans the filter, enabling it to be used much longer or for larger batches than equivalent TFF filters.

[0009] The TFF or ATF techniques of the prior art are characterized by flow of the cell media through a hollow fiber membrane, which is open on both ends. This allows the retentate including the cells to pass through the inner lumen of the hollow filter from the bioreactor, then back into the bioreactor, whereas the permeate can pass the filter membrane and can be separated from the retentate and discarded or stored in another vessel. As cells pass the inner lumen of the hollow filter, fouling of the membrane can occur. In the case of TFF, membrane fouling is addressed by intermittently sending cell-free buffer through the system, or by increasing the ratio of retentate flow rate / permeate flow rate, which however has limits due to increased shear forces. In ATF, fouling is addressed by the alternating pressure, which is designed to remove cells attached to the membrane periodically. However, in conventional TFF or ATF, fouling is still a considerable problem that needs to be addressed.

[0010] As can be seen, in the conventional set up, the culture medium comprising the cells has to leave the controlled environment of a bioreactor to pass a filtration loop, where the permeate is separated from the retentate, and then is returned to the bioreactor. In other words, filtration happens outside of a bioreactor. This complicates the handling and operation of the bioreactor / filtration system, increases the footprint, might not be optimal for the cells if the residence time outside of a bioreactor becomes too long and increases the costs. In addition, what limits use of perfusion in small scale systems like e.g. microbioreactors, is a need to draw a certain culture volume from a bioreactor into an external filtration loop which can be problematic given the already small culture volumes. Furthermore, fouling needs to be addressed by the techniques mentioned previously.

[0011] The development of a biopharmaceutical manufacturing process is labor and timeconsuming and usually requires multiple rounds of screening such as cell line selection, process condition definition or media optimization. This generates the need for reliably predictive small scale screening, for example to determine optimal culture conditions, since it’s not feasible to do all of those screens in the final production scale. The usage of parallelized microbioreactors can significantly accelerate the development and thereby increase the availability of medicines for patients. At the same time, the cost of goods can be lowered by developing high-yield-processes.

[0012] A general approach to scale down perfusion processes from large scale bioreactors to a microbioreactor is to use a perfusion mimic. The most common methods for this purpose are the cell settling method and the centrifugation method. Both methods have the disadvantage that the cells are not in suspension for a distinct period of time and therefore no constant supply of nutrients can be guaranteed. Furthermore, the level of dissolved oxygen and the pH level cannot be controlled during this period. This can lead to differences of the cell metabolism and thereby the predictivity of the scale-down model is reduced. As the purpose of the small scale screening is however to obtain reliable data, which can then be used in a large scale model, there is a need to develop a reliably predictive small scale screening. In addition, both the settling method and the centrifugation methods require frequent and lengthy interruptions of an otherwise well controlled cell cultivation in a bioreactor, making the process discontinuous, unlike a true perfusion process. Therefore, there is still a need for the provision of a filtration device, which can mimic existing large scale filtration applications.

[0013] This need was addressed by the present invention, which provides a cell retention device, which is characterized by comprising a hollow fiber membrane, which is closed at one end, providing a dead end filter. The cell retention device of the present invention allows separation of retentate and permeate without the need of a filtration loop requiring cells to leave the culture vessel, as the cells remain in the vessel comprising the cell retention device. Furthermore, as the cells remain within the culture vessel throughout the fermentation process, the culture parameters can be better controlled. The in situ filtration furthermore minimizes the dimensions needed for the microbioreactor, as the external filtration loop needed in conventional techniques is not necessary. As no cells can pass through the inner lumen of the hollow fiber, as its distal end is closed, fouling of the inner surface of the membrane can be avoided. Fouling of the outer surface can be easily prevented by stirring the culture medium which takes place in a bioreactor and / or by flushing the outer surface with fresh culture medium during the fermentation process. These advantages also provide the possibility to use the cell retention device in each of a plurality of microbioreactors in a system of microbioreactors, which can then be handled in parallel by a single operator. With the cell retention device of the present invention culture medium can be filtered out of a microbioreactor. The microbioreactor can be used for seed train cultures, inoculum train cultures or main fermentation (production phase). SUMMARY OF THE INVENTION

[0014] The present invention is concerned with a cell retention device as defined in the claims for use within a culture vessel of a microbioreactor. The present invention is also concerned with a microreactor comprising said cell retention device, and with a system comprising a plurality of microreactors each comprising said cell retention device. The present invention also relates to the use of the cell retention device of the invention for filtering culture medium out of a microbioreactor and to the use of the microbioreactor comprising the cell retention device of the invention for seed train culture, inoculum train culture or a main fermentation (production phase). The present invention further relates to a method of exchanging culture medium in a microbioreactor comprising the cell retention device of the invention.

[0015] The cell retention device of the present invention is suitable for use within a culture vessel of a microbioreactor and comprises a housing with a closed end and an open end, at least one hollow fiber filter supported within the housing and protruding with its open proximal end through the closed end of the housing. In some embodiments, the cell retention device also comprises an optional inlet port for introduction of fluid, preferably culture medium. The distal end of the at least one hollow fiber filter is arranged near or at the open end of the housing for being exposed to the outside of the housing, and the at least one hollow fiber filter comprises a closed front surface at its distal end. In embodiments comprising more than one hollow fiber filter, the distal end of each hollow fiber filter comprises a closed front surface at its distal end.

[0016] In one embodiment of the present invention at least a part of the housing of the cell retention device extends over the entire length of the hollow fiber filter.

[0017] Alternatively or additionally, in one embodiment of the present invention one housing segment at the open end of the housing the cell retention device protrudes further away from the closed end of the housing than another housing segment at the open end of the housing, the housing segments opposing each other. Thereby, the open end of the housing provides a window in the housing, for exposing the hollow fiber filter to the outside.

[0018] Alternatively or additionally, in one embodiment of the present invention the housing comprises a connector, preferably a Luer connector, at its closed end, for connection with the proximal end of the hollow fiber filter.

[0019] Alternatively or additionally, in one embodiment of the present invention the closed end of the housing is constituted by a sealing wall provided around the at least one hollow filter fiber, preferably wherein the sealing wall is made of adhesive. In the present invention, the housing is preferably substantially a tube-like body. Alternatively or additionally, the housing can be made of plastic material, preferably a transparent plastic material.

[0020] In the cell retention device of the present invention the closed front surface of the distal end of the hollow fiber filter can be sealed shut, preferably by an adhesive.

[0021] Furthermore, the hollow fiber filter can extend within the housing in a longitudinal manner. Furthermore, the hollow fiber filter can be arranged coaxially with the central longitudinal axis of the housing. In a preferred embodiment, the hollow fiber filter is a Tangential Flow Filtration (TFF) filter. A TFF filter is a filter that is suitable for use in a tangential flow filtration method. The TFF hollow fiber filter can for example be produced using polymers, such as polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polypropylene (PP), polyethersulfone (PES), nylon / polyamide (NYL), cellulose acetate (CA), cellulose nitrate (CN), regenerated cellulose (RC), polyvinylpyrrolidone (PEV), or combinations thereof as known in the art. The polymers can be surface modified as known in the art. The TFF hollow fiber filter can comprise inorganic aluminum oxide (Anopore (ANP)) or glass microfiber / glass fiber (GMF / GF). In a preferred embodiment, the TFF hollow fiber filter is produced using polyethersulfone (PES) and / or modified polyethersulfone (mPES).

[0022] Furthermore, the hollow fiber filter can be supported within the housing by a sealing wall. Preferably, the sealing wall can be arranged within the housing near or at a window in the housing, for exposing the hollow fiber filter to the outside. In a preferred embodiment, the sealing wall is made of an adhesive.

[0023] In a preferred embodiment of the invention, the hollow fiber filter can have an inside diameter ranging from 0.5 mm to 1 mm. Furthermore, the hollow fiber filter can comprise hollow fiber filter material providing an average pore size ranging from 0.2 pm to 2.0 pm, preferably 0.2 pm to 0.65 pm. The pore size can be adjusted by the skilled person depending on the molecule sizes to be restricted.

[0024] The cell retention device of the present invention comprising at least one hollow fiber filter preferably comprises at least two hollow fiber filters, which are supported within the housing and are protruding with their open proximal ends through the closed end of the housing. The hollow fiber filters are preferably arranged coaxially with each other and with the central longitudinal axis of the housing.

[0025] In a preferred embodiment, the cell retention device of the present invention is for perfusion of mammalian cell suspension culture.

[0026] The cell retention device of the present invention is for use within a culture vessel of a microbioreactor. The present invention is therefore also concerned with a microbioreactor comprising a culture vessel having a working volume of about 15 ml or less, a cell retention device according to the invention; a mixing component for mixing a content of the culture vessel; and an inlet port for introduction of culture medium components and reagents to the culture vessel. The cell retention device is arranged at least in part within the culture vessel, and the microbioreactor is configured to allow removal of culture medium from the culture vessel through the at least one hollow fiber filter of the cell retention device, preferably in a continuous manner.

[0027] In a preferred embodiment of the microbioreactor of the present invention, one housing segment at the open end of the housing of the cell retention device protrudes further away from the closed end of the housing than another housing segment at the open end of the housing, the housing segments opposing each other. The open end of the housing thereby provides a window in the housing, for exposing the hollow fiber filter to the culture vessel, and the cell retention device is arranged in such a way that the window in the housing is facing away from the bioreactor’s mixing component. In this embodiment, the hollow fiber filter is protected by the housing from a direct impact of flow effected by the mixing component. In other embodiments, the window can also be directed towards the mixing component.

[0028] The microbioreactor of the present invention can further comprise a source of negative pressure, preferably in the form of a pump, connected to the proximal end of the hollow fiber filter, for drawing culture medium out of the culture vessel through the hollow fiber filter; an inoculation port for inoculation with cells to be cultured; an addition port for addition of antifoam and / or base; a port for extraction of cultured cells and / or supernatant; a gassing port system for addition of O2, N2 and / or CO2; a means for detection of flow rate; an internal sensor for detection of dissolved oxygen within the culture vessel; an internal sensor for measurement of pH within the culture vessel; and / or a temperature regulating element for heating or cooling a content of the culture vessel.

[0029] In the microbioreactor of the present invention, the inlet port can be provided in the housing of the hollow fiber filter of the cell retention device. Furthermore, the mixing component can comprise a stirrer. Preferably, the stirrer can be arranged on a shaft protruding into the culture vessel. In preferred embodiments, the stirrer can be a pitched- blade stirrer or a Rushton turbine. Furthermore, in the microbioreactor of the present invention, at least a portion of the inner surface of the culture vessel can be surface-modified to resist adherence of cells and / or proteins.

[0030] The present invention is also concerned with the use of the cell retention device of the present invention for filtering culture medium out of a microbioreactor of the present invention. The present invention is also concerned with the use of a microbioreactor of the present invention for seed train culture, inoculum train culture or a main fermentation (production phase).

[0031] The present invention is also concerned with a method of exchanging culture medium in a microbioreactor of the present invention by means of a cell retention device of the present invention, comprising the steps of a) cultivating cells within the culture vessel by means of the culture medium; and b) removing culture medium through the at least one hollow fiber filter of the cell retention device; c) replacing removed culture medium with fresh medium through the inlet port of the microbioreactor or of the cell retention device, either continuously at a constant flow rate, or intermittently with a bolus injection of fresh culture medium.

[0032] In a preferred embodiment of the method of exchanging culture medium of the present invention, removing culture medium through the at least one hollow fiber filter of the cell retention device can be a continuous process. Alternatively or additionally, the process of replacing removed culture medium with fresh medium through the inlet port of the microbioreactor or of the cell retention device can be a continuous process with a constant flow rate of culture medium into the culture vessel; or the process of replacing removed culture medium with fresh medium through the inlet port of the microbioreactor or of the cell retention device can be carried out intermittently with a bolus injection of fresh culture medium. In a preferred embodiment of the method of the invention, steps a) to c) can be repeated.

[0033] The present invention is also concerned with a system of at least two microbioreactors of the present invention, wherein the system is configured to operate the microbioreactors in parallel.

[0034] The system of the present invention can for example comprise 12, 24, or 48 microbioreactors of the present invention. The system can further comprise a holder configured to hold each microbioreactor in place during operation; a pumping system, preferably wherein the pumping system is at least in part pressure driven; at least one filling level sensor for each microbioreactor for monitoring the culture medium level in each of the microbioreactors; a fresh culture medium reservoir in fluid communication with each of the microbioreactors, for addition of fresh culture medium into each culture vessel; a waste reservoir in fluid communication with each of the microbioreactors for retention of culture medium removed from each of the microbioreactors; a heating / cooling system; a control unit configured to effect addition of fresh culture medium, optionally in response to a signal obtained from a filling level sensor, and / or to maintain a steady temperature across the system by controlling a heating / cooling system. Advantageously, the system of the present invention can be configured to be controlled by a single operator.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] In the following, the present invention is described in detail. The features of the present invention are described in individual paragraphs. This, however, does not mean that a feature described in a paragraph stands isolated from a feature or features described in other paragraphs. Rather, a feature described in a paragraph can be combined with a feature or features described in other paragraphs.

[0037] The term “comprise / s / ing”, as used herein, is meant to include or encompass the disclosed features and further features which are not specifically mentioned. The term “comprise / es / ing” is also meant in the sense of “consist / s / ing of” the indicated features, thus not including further features except the indicated features. Thus, the subject-matter of the present invention may be characterized by additional features in addition to the features as indicated.

[0038] As used herein and also in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Similarly, the words "comprise", "contain" and "encompass" are to be interpreted inclusively rather than exclusively; that is to say, in the sense of “including, but not limited to”. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The terms "plurality", “multiple” or “multitude” refer to two or more, i.e. 2 or >2, with integer multiples, wherein the terms “single” or “sole” refer to one, i.e. =1. Furthermore, the term “at least one” is to be understood as one or more, i.e. 1 or >1 , also with integer multiples. Accordingly, words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,”, “previously” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0039] Furthermore, certain terms are used for reasons of convenience and are not intended to limit the invention. The terms “right”, “left”, “up”, “down”, “under" and “above" refer to directions in the figures. The terminology comprises the explicitly mentioned terms as well as their derivations and terms with a similar meaning. Also, spatially relative terms, such as "beneath", "below", "lower", "above", "upper", "proximal", "distal", and the like, may be used to describe one element's or feature's relationship to another element or feature as illustrated in the figures. These spatially relative terms are intended to encompass different positions and orientations of the devices in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be "above" or "over" the other elements or features. Thus, the exemplary term "below" can encompass both positions and orientations of above and below. The devices may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein interpreted accordingly. Likewise, descriptions of movement along and around various axes include various special device positions and orientations.

[0040] With the cell retention device of the present invention, it becomes possible to filter material comprised in a culture vessel, in which the cell retention device is placed, through the membrane of the hollow fiber filter into the inner lumen of the hollow fiber filter, and thereby material can be removed from the culture vessel. The hollow filter membrane has an open proximal end, which protrudes through the closed end of the housing. The permeate, which is the material having passed the filter membrane, can be removed using the open proximal end of the hollow fiber membrane as on outlet port. Cells comprised in the culture vessel cannot pass through the hollow filter membrane.

[0041] Furthermore, cells cannot enter the inner lumen via the distal end of the hollow fiber filter, as the distal end is closed. Therefore, a fouling of the inner surface of the membrane by attachment of cells can be avoided. Another advantage of having a closed distal end of the hollow fiber filter is that the fermentation culture can remain within the same culture vessel during fermentation, and does not need to pass a filtration loop, as is the case in traditional ATF or TFF devices.

[0042] With the cell retention device of the present invention it is therefore possible to cost- efficiently and reliably provide the culture vessel with stable culture conditions by removing culture medium via the cell retention device and by replenishing with fresh culture medium via the inlet port. The efficacy of the hollow fiber filter is not compromised by fouling, as cells cannot enter the inner lumen. Fouling of the outer surface of the hollow fiber filter can be easily prevented by the convection provided by a mixing component in the culture vessel, and / or by the flow of fresh culture medium provided via the inlet port.

[0043] In one embodiment of cell retention device of the present invention, the closed end of the housing is constituted by a sealing wall. The sealing wall separates the closed end from the open end of the housing, and provides a further barrier for material from the culture vessel. In another embodiment, the sealing wall is not present. In this embodiment, material from the culture vessel can enter the space provided between the inner wall of the housing and the outer surface of the hollow fiber filter. Furthermore, material introduced via the inlet port can enter the space provided between the inner wall of the housing and the outer surface of the hollow fiber filter as well. If a sealing wall is present at the closed end of the housing, only the segment of the hollow filter fiber which is exposed to the culture vessel via the window in the housing provides an accessible interface for removing material from the culture vessel. A mixing component can be arranged within the culture vessel to provide turbulence or convection at the window to further decrease the risk of fouling. Furthermore, when a sealing wall is present, and an inlet port is provided in the cell retention device at a position where the material introduced via the inlet port enters the space between the inner wall of the housing and the outer surface of the hollow fiber filter, material, such as buffer or culture medium can be introduced via the inlet port if the membrane needs to be flushed. In this embodiment, fresh material can be introduced into the culture vessel via another inlet port in the microbioreactor.

[0044] The distal end of the hollow fiber filter comprised in the cell retention device of the present invention is arranged near or at the open end of the housing. The open end of the housing provides the window to the culture vessel, and therefore the main interface for material transport via the hollow fiber filter. As described above, a mixing component can provide agitation of the material inside the culture vessel, which can provide aeration and homogenous distribution, but also prevents attachment of material to the outer surface of the hollow fiber filter, and thereby fouling. If the material of the hollow fiber filter is delicate and prone to be disturbed by the movement provided by the mixing component, the window of the housing can be arranged in a way that the window and the mixing component are opposite to each other. In this setup, a direct flow from the mixing component to the exposed outer surface of the hollow fiber filter is blocked by the housing (see, e.g. Figures 5 and 7).

[0045] The hollow fiber filter can comprise hollow fiber filter material as known in the art, and can have dimensions as known in the art. For example, the inner lumen can have a diameter ranging from 0.5 mm to 1 mm. The hollow fiber filter material can have an average pore size ranging from 0.2 pm to 2.0 pm, and can be adjusted depending on the size of the particles that need to be able to pass the pores. Preferred average pore sizes are 0,2 pm and 0.65 pm. The average pore size is determined as is known in the art, and usually known for a hollow fiber material.

[0046] The cell retention device of the present invention comprises at least one hollow fiber filter. To increase material flow, more than one hollow fiber filter can be positioned in the cell retention device, preferably in parallel. Preferably, at least two hollow fiber filters are present in a cell retention device of the present invention (see e.g. Figures 1B, 2B, 3B, 4B).

[0047] As disclosed above, the cell retention device of the present invention is for use within a culture vessel of a microbioreactor. For the purpose of the present invention, a microbioreactor is defined as comprising a culture vessel having a working volume of about 15 ml or less. A “working volume”, as understood by the skilled person, is not referring to the total volume of the vessel, but to the maximum amount of volume filled with any material during operation of the reactor, such as cell culture medium and cells. The microbioreactor also comprises a head space, which is a term to describe the empty space in the fermentation tanks. That space should not be full of any material, as it’s the safe margin of the tank. Pressured tanks, for example, must have enough head space to ensure security.

[0048] The microbioreactor of the invention also comprises at least one inlet port for introduction of culture medium components and reagents to the culture vessel. Exemplary reagents used in fermentation are correction agents such as antifoam, base, glucose and the like. The inlet port can be comprised in the cell retention device, or it can be separated from the cell retention device. In the latter case, such as in the embodiments of Figures 5 and 6, the inlet port can be positioned, for example in a wall of the culture vessel (not shown in Figures 5 and 6).

[0049] The microbioreactor of the invention can also comprise further components, which are usual for a fermentation system. For example, a source of negative pressure can be provided, preferably in the form of a pump, connected to the proximal end of the hollow fiber filter, for drawing culture medium out of the culture vessel through the hollow fiber filter. Further components can be an inoculation port for inoculation with cells to be cultured, an addition port for addition of antifoam and / or base; a port for extraction of cultured cells and / or supernatant; a gassing port system for addition of O2, N2 and / or CO2; a means for detection of flow rate; an internal sensor for detection of dissolved oxygen within the culture vessel; an internal sensor for measurement of pH within the culture vessel; and / or a temperature regulating element for heating or cooling a content of the culture vessel

[0050] These components can be controlled by a control unit as is known in the art.

[0051] The control unit of the system of the invention can also control any kind of actuation or monitoring of the above described system and its components, wherein the term “control unit” as used herein encompasses any physical or virtual processing device, such as a CPU or the like, which can also control the entire microbioreactor or even an entire system of microbioreactors comprising one or more laboratory instruments in a way that workflow(s) and workflow step(s) are conducted. The control unit may, for example, carry different kinds of application software and instruct the automated processing system or a specific instrument or device thereof to conduct pre-analytical, post analytical and analytical workflow(s) / workflow step(s). The control unit may receive information from a data management unit regarding which steps need to be performed with a certain sample. Further, the control unit might be integral with a data management unit, may be comprised by a server computer and / or be part of one instrument or even distributed across multiple instruments of the automated processing system. The control unit may, for instance, be embodied as a programmable logic controller running a computer-readable program provided with instructions to perform operations. Here, in order to receive such instructions by a user, a user interface can additionally be provided, wherein the term “user interface” as used herein encompasses any suitable piece of application software and / or hardware for interactions between an operator and a machine, including but not limited to a graphical user interface for receiving as input a command from an operator and also to provide feedback and convey information thereto. Also, a system / device may expose several user interfaces to serve different kinds of users I operators.

[0052] The cell retention device of the present invention can be used for filtering culture medium out of a microbioreactor comprising the cell retention device.

[0053] The microbioreactor comprising the cell retention device of the present invention can also be used for seed train culture. As known in the art, the seed train is the scaling of the culture from a small volume of cells in a culture vessel to a larger volume of cells that are used to inoculate the main production reactor at the required cell density. The microbioreactor comprising the cell retention device of the present invention can also be used for inoculum train culture. In the present invention, the first cell culture fermentation processes after thawing of cells are termed “seed train” culture, wherein subsequent fermentation processes using the cells resulting from the seed train culture are termed “inoculum train culture”. The microbioreactor comprising the cell retention device of the present invention can also be used for main fermentation (production phase).

[0054] As described above, the cell retention device of the present invention can be used in a method of exchanging culture medium in a microbioreactor. Therefore, the present invention is also concerned with a method of exchanging culture medium in a microbioreactor of the present invention by means of a cell retention device of the present invention. During cultivation of cells within the culture vessel, the nutrients in culture medium will decrease and metabolic by-products, often with toxic or other undesired effects on cells, will accumulate. Therefore, the spent medium needs to be removed, and replaced by fresh medium. In the present invention, this is accomplished by removing culture medium through the at least one hollow fiber filter of the cell retention device and by replacing removed culture medium with fresh medium through the inlet port of the microbioreactor or of the cell retention device. The replacing step can be either continuously at a constant flow rate, or intermittently with a bolus injection of fresh culture medium.

[0055] In a preferred embodiment of the method of exchanging culture medium of the present invention, removing culture medium through the at least one hollow fiber filter of the cell retention device can be a continuous process. In a preferred embodiment of the method of the invention, the steps of cultivating cells, removing culture medium, and replacing removed medium with fresh medium can be repeated, for example until a desired cell density is reached in the culture vessel.

[0056] The present invention is also concerned with a system of at least two microbioreactors of the present invention, wherein the system is configured to operate the microbioreactors in parallel. Due to the compact design of the microbioreactor of the present invention, where the cultivated cells remain in the culture vessel during the whole fermentation process, a parallel operation of a plurality of microbioreactors can be efficiently achieved. The medium can be removed via the hollow fiber filter of the cell retention device, for example with a pumping system, which achieves a constant flow rate of medium through the membrane of the hollow fiber filter, into the inner lumen of the hollow fiber filter, and then via the proximal end out of the microbioreactor. Fresh medium can be replaced via the inlet port at a constant rate, or if desired, intermittently, for example as bolus injection. The constant flow into the microbioreactor, and the bolus injection as well, can be used to flush the outer surface of the hollow fiber filter, where it is exposed. Additionally, the convection or turbulence introduced by the mixing component can also prevent attachment of material to the outer surface of the hollow fiber filter. Thereby, the risk of fouling is further reduced.

[0057] The system of the present invention can for example comprise 12, 24, or 48 microbioreactors of the present invention. The system can further comprise a holder configured to hold each microbioreactor in place during operation; a pumping system, preferably wherein the pumping system is at least in part pressure driven; at least one filling level sensor for a microbioreactor for monitoring the culture medium level in the microbioreactor; a fresh culture medium reservoir in fluid communication with each of the microbioreactors, for addition of fresh culture medium into each culture vessel; a waste reservoir in fluid communication with each of the microbioreactors for retention of culture medium removed from each of the microbioreactors; a heating / cooling system; a control unit configured to effect addition of fresh culture medium, optionally in response to a signal obtained from a filling level sensor, and / or to maintain a steady temperature across the system by controlling a heating / cooling system.

[0058] Advantageously, the system of the present invention can be configured to be controlled by a single operator. This allows efficient and fast performance of test series. For example, in a system of 12 microbioreactors, culture parameters can be varied, and the best combination of parameters can be found out quickly. Furthermore, a plurality of seed train cultures or inoculum train cultures can be obtained in parallel, efficiently and easily, which makes it easier to determine the optimal culture conditions to be later used in large-scale production.

[0059] To avoid repetition in the figures and the descriptions of the various aspects and illustrative embodiments, it should be understood that many features are common to many aspects and embodiments. The description of specific embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. Specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure as defined by the appended claims. Omission of an aspect from a description or figure does not imply that the aspect is missing from embodiments that incorporate that aspect. Instead, the aspect may have been omitted for clarity and to avoid prolix description. In this context, the following applies to the rest of this description: If, in order to clarify the drawings, a figure contains reference signs which are not explained in the directly associated part of the description, then it is referred to previous or following description sections. Further, for the reason of lucidity, if in a section of a drawing not all features of a part are provided with reference signs, it is referred to other sections of the same drawing. Like numbers in two or more figures represent the same or similar elements.

[0060] The invention is further explained by the attached figures and examples, which are intended to illustrate, but not to limit the present invention.

[0061] FIGURES

[0062] Figure 1 shows one exemplary embodiment of the cell retention device of the present invention. Figure 1A shows a general view of the cell retention device. Figure 1 B shows top view of the cell retention device. Figure 1C shows a cross section of the cell retention device (1) along the line from A to A as seen in Figure 1 B. The housing (2) comprises a closed end (3) and an open end (4). A hollow fiber filter (5) is arranged coaxially with the central longitudinal axis of the housing. As can be seen from Figure 1 B, the cell retention device comprises two hollow fiber filter arranged in parallel. The distal end (7) of the hollow fiber filter is closed. As can also be seen, the hollow fiber filter is exposed to the outside at the window (8), defined by the open end of the housing. The cell retention device of Figure 1 further comprises a sealing wall (9) separating the closed end of the housing from the open end of the housing, and thereby restricting access into the device from the outside via the open end. The cell retention device of Figure 1 further comprises an inlet port (6) for the introduction of fluid, such as culture medium. As the cell retention device of Figure 1 comprises a sealing wall (9), culture medium introduced via inlet port (6) can be used to flush the outer surface of the hollow fiber filter (5), if desired.

[0063] Figure 2 shows one exemplary embodiment of the cell retention device of the present invention. Figure 2A shows a general view of the cell retention device. Figure 2B shows top view of the cell retention device. Figure 2C shows a cross section of the cell retention device (1) along the line from A to A as seen in Figure 2B. The housing (2) comprises a closed end (3) and an open end (4). A hollow fiber filter (5) is arranged coaxially with the central longitudinal axis of the housing. As can be seen from Figure 2B, the cell retention device comprises two hollow fiber filter arranged in parallel. The distal end (7) of the hollow fiber filter is closed. As can also be seen, the hollow fiber filter is exposed to the outside at the window (8), defined by the open end of the housing. The cell retention device of Figure 2 further comprises a sealing wall (9) separating the closed end of the housing from the open end of the housing, and thereby restricting access into the device from the outside via the open end. The cell retention device of Figure 2 further comprises an inlet port (6) for the introduction of fluid, such as culture medium. As the cell retention device of Figure 2 comprises a sealing wall (9), culture medium introduced via inlet port (6) can be used to flush the outer surface of the hollow fiber filter (5).

[0064] Figure 3 shows one exemplary embodiment of the cell retention device of the present invention. Figure 3A shows a general view of the cell retention device. Figure 3B shows top view of the cell retention device. Figure 3C shows a cross section of the cell retention device (1) along the line from A to A as seen in Figure 3B. The housing (2) comprises a closed end (3) and an open end (4). A hollow fiber filter (5) is arranged coaxially with the central longitudinal axis of the housing. As can be seen from Figure 3B, the cell retention device comprises two hollow fiber filter arranged in parallel. The distal end (7) of the hollow fiber filter is closed. As can also be seen, the hollow fiber filter is exposed to the outside at the window (8), defined by the open end of the housing. The cell retention device of Figure 3 further comprises an inlet port (6) for the introduction of fluid, such as culture medium. As the cell retention device of Figure 3 does not comprise a sealing wall, culture medium introduced via inlet port (6) can be used to flush the outer surface of the hollow fiber filter (5), and it can also be introduced into the culture vessel, into which the cell retention device (1) can be placed.

[0065] Figure 4 shows one exemplary embodiment of the cell retention device of the present invention. Figure 4A shows a general view of the cell retention device. Figure 4B shows top view of the cell retention device. Figure 4C shows a cross section of the cell retention device (1) along the line from A to A as seen in Figure 4B. The housing (2) comprises a closed end (3) and an open end (4). A hollow fiber filter (5) is arranged coaxially with the central longitudinal axis of the housing. As can be seen from Figure 4B, the cell retention device comprises two hollow fiber filter arranged in parallel. The distal end (7) of the hollow fiber filter is closed. As can also be seen, the hollow fiber filter is exposed to the outside at the window (8), defined by the open end of the housing. The cell retention device of Figure 4 further comprises an inlet port (6) for the introduction of fluid, such as culture medium. As the cell retention device of Figure 4 does not comprise a sealing wall, culture medium introduced via inlet port (6) can be used to flush the outer surface of the hollow fiber filter (5), and it can also be introduced into the culture vessel, into which the cell retention device (1) can be placed.

[0066] Figure 5 shows a cross sectional view of a microbioreactor (10) of the present invention comprising the cell retention device (1) of Figure 1. The cell retention device is placed in a culture vessel (11) further comprising a mixing component (12). As can be seen, the mixing component is arranged within the culture vessel opposite to the window in the housing. For introduction of fresh culture medium, the microbioreactor (10) comprises an inlet port (not shown).

[0067] Figure 6 shows a cross sectional view of a microbioreactor (10) of the present invention comprising the cell retention device (1) of Figure 2. The cell retention device is placed in a culture vessel (11) further comprising a mixing component (12). As can be seen, the window in the housing is directed towards the mixing component. For introduction of fresh culture medium, the microbioreactor (10) comprises an inlet port (not shown).

[0068] Figure 7 shows a cross sectional view of a microbioreactor (10) of the present invention comprising the cell retention device (1) of Figure 3. The cell retention device is placed in a culture vessel (11) further comprising a mixing component (12). As can be seen, the mixing component is arranged within the culture vessel opposite to the window in the housing.

[0069] Figure 8 shows a cross sectional view of a microbioreactor (10) of the present invention comprising the cell retention device (1) of Figure 4. The cell retention device is placed in a culture vessel (11) further comprising a mixing component (12). As can be seen, the window in the housing is directed towards the mixing component.

[0070] The following examples are intended to illustrate various specific embodiments of the present invention. As such, the specific modifications as discussed hereinafter are not to be construed as limitations on the scope of the present invention. It will be apparent to the person skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present invention, and it is thus to be understood that such equivalent embodiments are to be included herein. Further aspects and advantages of the present invention will become apparent from the following description of particular embodiments illustrated in the figures. EXAMPLES

[0071] Example 1 - Cell retention device of Figure 1 and microbioreactor of Figure 5

[0072] As presented above, the general approach to scale down perfusion processes from large scale bioreactors to a microbioreactor is to use a perfusion mimic. The most common methods for this purpose are the cell settling method and the centrifugation method. Both methods have the disadvantage that the cells are not in suspension for a distinct period of time and therefore no constant supply of nutrients can be guaranteed. Furthermore, the level of dissolved oxygen and the pH level cannot be controlled during this period. This can lead to differences of the cell metabolism and thereby the predictivity of the scale-down model is reduced. With the microbioreactor (10) shown in Figure 5, this limitation can be overcome. The cell retention device (1) is about 10 cm long in total and placed in the microbioreactor to ensure a in situ cell separation. The device extends about 5.5 cm into the microbioreactor and the outer diameter of the housing is about 0.46 cm. The microbioreactor can be the commercially available microbioreactor “Ambr® 15” (Sartorius) with external dimensions of 3 x 1.7 x 6 cm (length x depth x height without lid) and a recommended working volume between 10 and 15 mL. The sealing wall of the device ensures that the cells remain in the mixed microbioreactor. The cell free spent media can be removed through the hollow fiber filter which is exposed by the window in the housing. The window is 2 cm long and the rest of the housing supports the filter to avoid bending of the flexible fiber. The orientation of the window points away from the stirrer to avoid direct exposure of the filter to the flow provided by the stirrer, while still exposing the filter to a certain indirect flow segment of the stirred microbioreactor. This flow reduces filter fouling. The spent media is removed continuously at a certain flowrate, which depends on the desired microbioreactor volumes per day. To keep the microbioreactor volume constant, fresh media is added at the same rate into the microbioreactor via an external port (not shown in figure 5). The addition and the removal is performed by two separate syringe pumps, which are connected to the cell retention device and the external port via tubings. The cell retention device can be inserted before or after the inoculation of the microbioreactors with cells. A typical experiment could be as follows. The microbioreactor is first inoculated with cells and, after an initial growth phase, the cell retention device is used. An ideal working volume could be 13 mL. After the batch phase, spent medium is removed continuously via a pump and fresh medium is added at the same rate. For this purpose, an exchange rate that increases with increasing cell density can be selected. The retentate and the permeate can be analyzed to calculate typical cell culture parameters such as consumption or growth rates. After the desired cultivation time, the perfusion can be stopped and the cells can be harvested. Example 2 - Cell retention device of Figure 2 and microbioreactor of Figure 6

[0073] With the microbioreactor (10) shown in Figure 6, the conventional limits of perfusion mimic as described above, can be overcome. The cell retention device is about 10 cm long in total and placed in the microbioreactor to ensure a in situ cell separation. The device extends about 5.5 cm into the microbioreactor and the outer diameter of the housing is about 0.46 cm. The microbioreactor can be the commercially available microbioreactor “Ambr® 15” (Sartorius) with external dimensions of 3 x 1.7 x 6 cm (length x depth x height without lid) and a recommended working volume between 10 and 15 mL. The sealing wall of the device ensures that the cells remain in the mixed microbioreactor. The cell free spent media can be removed through the hollow fiber filter, which is exposed by the window in the housing. The window is 2 cm long and the rest of the housing supports the filter to avoid bending of the flexible fiber. The orientation of the window points towards the stirrer to expose the filter to a certain flow segment of the stirred bioreactor. This flow reduces the risk of filter fouling. The spent media is removed continuously at a certain flowrate, which depends on the desired vessel volumes per day. To keep the microbioreactor volume constant, fresh media is added at the same rate into the bioreactor via an external port (not shown in Figure 6). The addition and the removal is performed by two separate syringe pumps, which are connected to the cell retention device and the external port via tubings. The cell retention device can be inserted before or after the inoculation of the microbioreactors with cells. A typical experiment could be as follows. The microbioreactor is first inoculated with cells and, after an initial growth phase, the cell retention device is used. An ideal working volume could be 13mL. After the batch phase, used medium is removed continuously via a pump and fresh medium is added at the same rate. For this purpose, an exchange rate that increases with increasing cell density can be selected. The retentate and the permeate can be analyzed to calculate typical cell culture parameters such as consumption or growth rates. After the desired cultivation time, the perfusion can be stopped and the cells can be harvested.

[0074] Example 3 - Cell retention device of Figure 3 and microbioreactor of Figure 7

[0075] With the microbioreactor (10) shown in Figure 7, the conventional limits of perfusion mimic as described above, can be overcome. The cell retention device is about 10 cm long in total and placed in the microbioreactor to ensure a in situ cell separation. The device extends about 5.5 cm into the microbioreactor and the outer diameter of the housing is about 0.46 cm. The microbioreactor can be the commercially available microbioreactor “Ambr® 15” (Sartorius) with external dimensions of 3 x 1 .7 x 6 cm (length x depth x height without lid) and a recommended working volume between 10 and 15 mL. Unlike the cell retention device of Figure 1 or Figure 2, the cell retention device of Figure 3 does not have a sealing wall at the distal end. As a result, the entire length of the hollow fiber can be used for the extraction of spent media. The window at the distal end is 2 cm long. The orientation of the window points away from the stirrer to avoid direct exposure of the filter to the flow provided by the stirrer, while still exposing the filter to a certain indirect flow segment of the stirred microbioreactor. This flow reduces the risk of filter fouling. The spent media is removed continuously at a certain flowrate, which depends on the desired microbioreactor volumes per day. Fresh media is added into the microbioreactor via the inlet port (6). To ensure that the cells that are drawn between the filter housing and the hollow fiber, are returned to the mixed part of the microbioreactor, fresh medium can be added in batches and at a significantly higher rate. This reduces the risk of filter blockage, since the part of the filter that is not exposed to the turbulent flow in the reactor is flushed by fresh media, which flows into the microbioreactor. Depending on the desired removal rate, the intervals can be selected. A possible mode would be a continuous removal and a fresh medium addition every 3 h, with the addition taking only 30 seconds (i.e. bolus injection). The amount of removed media and added media is identical. The addition and the removal is performed by two separate syringe pumps, which are connected to the cell retention device and the external port via tubings. The cell retention device can be inserted before or after the inoculation of the microbioreactors with cells. A typical experiment could be as follows. The microbioreactor is first inoculated with cells and, after an initial growth phase, the cell retention device is used. An ideal working volume could be 13mL. After the batch phase, used medium is removed continuously via a pump and fresh medium is added in the described batch mode. For this purpose, an exchange rate that increases with increasing cell density can be selected. The retentate and the permeate can be analyzed to calculate typical cell culture parameters such as consumption or growth rates. After the desired cultivation time, the perfusion can be stopped and the cells can be harvested.

[0076] Example 4 - Cell retention device of Figure 4 and microbioreactor of Figure 8

[0077] With the microbioreactor (10) shown in Figure 8, the conventional limits of perfusion mimic as described above, can be overcome. The cell retention device is about 10 cm long in total and placed in the microbioreactor to ensure a in situ cell separation. The device extends about 5.5 cm into the microbioreactor and the outer diameter of the housing is about 0.46 cm. The microbioreactor can be the commercially available microbioreactor “Ambr® 15” (Sartorius) with external dimensions of 3 x 1 .7 x 6 cm (length x depth x height without lid) and a recommended working volume between 10 and 15 mL. Unlike the cell retention device of Figure 1 or Figure 2, the cell retention device of Figure 4 does not have a sealing wall at the distal end. As a result, the entire length of the hollow fiber can be used for the extraction of spent media. The window at the distal end is 2 cm long and the orientation of the window points towards the stirrer to expose the filter to a certain flow segment of the stirred bioreactor. This flow reduces the risk of filter fouling. The spent media is removed continuously at a certain flowrate, which depends on the desired microbioreactor volumes per day. Fresh media is added into the microbioreactor via the inlet port (6). To ensure that the cells that are drawn between the filter housing and the hollow fiber are returned to the mixed part of the bio microbioreactor reactor, fresh medium can be added in batches and at a significantly higher rate. This reduces the risk of filter blockage, since the part of the filter that is not exposed to the turbulent flow in the microbioreactor is flushed by fresh media, which flows into the microbioreactor. Depending on the desired removal rate, the intervals can be selected. A possible mode would be a continuous removal and a fresh medium addition every 3 h, with the addition taking only 30 seconds (i.e. a bolus injection). The amount of removed media and added media is identical. The addition and the removal is performed by two separate syringe pumps, which are connected to the cell retention device and the external port via tubings. The cell retention device can be inserted before or after the inoculation of the microbioreactors with cells. A typical experiment could be as follows. The microbioreactor is first inoculated with cells and, after an initial growth phase, the cell retention device is used. An ideal working volume could be 13mL. After the batch phase, used medium is removed continuously via a pump and fresh medium is added in the described batch mode. For this purpose, an exchange rate that increases with increasing cell density can be selected. The retentate and the permeate can be analyzed to calculate typical cell culture parameters such as consumption or growth rates. After the desired cultivation time, the perfusion can be stopped and the cells can be harvested

[0078] Example 5 - System of microbioreactors operated in parallel

[0079] Examples 1-4 generally show how the cell retention device can be used in a microbioreactor. The parallelization of such a reactor allows an operator to look after a plurality of reactors simultaneously. This increases the throughput and thus opens up more possibilities of use. Typical use cases would be the screening of cell lines for biopharmaceutical production. A typical application is the screen of cell lines that produce biopharmaceutical molecules. In addition to the screening of different cell lines, a parallelized system can also be used for media and process development.

[0080] An apparatus could consist, for example, of 24 or 48 microbioreactor, which are controlled by one or more control computers. The control computer checks the process parameters of the microbioreactor, such as the pH value, temperature, stirrer speed, and media exchange rates. The filling level and the foam level can be visually checked by the operator or via a suitable sensor system, such as a camera with algorithm image processing software.

[0081] While the current invention has been described in relation to its specific embodiments, it is to be understood that this description is for illustrative purposes only. Accordingly, it is intended that the invention be limited only by the scope of the claims appended hereto.

[0082] REFERENCE LIST

[0083] 1 : Cell retention device

[0084] 2: Housing 3: Closed end of housing

[0085] 4: Open end of housing

[0086] 5: Hollow fiber filter

[0087] 6: Inlet port

[0088] 7: Distal end of the hollow fiber filter 8: Window in the housing

[0089] 9: Sealing wall

[0090] 10: Microbioreactor

[0091] 11 : Culture vessel

[0092] 12: Mixing component

Claims

CLAIMS1. A cell retention device (1) for use within a culture vessel of a microbioreactor, the cell retention device comprising a housing (2) with a closed end (3) and an open end (4) , at least one hollow fiber filter (5) supported within the housing and protruding with its open proximal end through the closed end of the housing, and an optional inlet port (6) for introduction of fluid, preferably culture medium, wherein a distal end (7) of the at least one hollow fiber filter is arranged near or at the open end of the housing for being exposed to the outside of the housing, and the at least one hollow fiber filter comprises a closed front surface at its distal end.

2. The cell retention device according to claim 1 , wherein2-1) at least a part of the housing extends over the entire length of the hollow fiber filter;2-2) one housing segment at the open end of the housing protrudes further away from the closed end of the housing than another housing segment at the open end of the housing, the housing segments opposing each other, the open end of the housing thereby providing a window (8) in the housing, for exposing the hollow fiber filter to the outside;2-3) the housing comprises a connector, preferably a Luer connector, at its closed end, for connection with the proximal end of the hollow fiber filter;2-4) the closed end of the housing is constituted by a sealing wall (9) provided around the at least one hollow filter fiber, preferably wherein the sealing wall is made of adhesive;2-5) the housing is substantially a tube-like body; and / or2-6) the housing is made of plastic material, preferably a transparent plastic material.

3. The cell retention device according to claim 1 or claim 2, wherein3-1) the closed front surface of the distal end of the hollow fiber filter is sealed shut, preferably by an adhesive;3-2) the hollow fiber filter extends within the housing in a longitudinal manner;3-3) the hollow fiber filter is arranged coaxially with the central longitudinal axis of the housing;3-4) the hollow fiber filter is a Tangential Flow Filtration, or TFF, filter;3-5) the hollow fiber filter is supported within the housing by a sealing wall, preferably wherein the sealing wall is arranged within the housing near or at a window in the housing, for exposing the hollow fiber filter to the outside, further preferably wherein the sealing wall is made of an adhesive;3-6) the hollow fiber filter has an inside diameter ranging from 0.5 mm to 1 mm;3-7) the hollow fiber filter comprises hollow fiber filter material providing an average pore size ranging from 0.2 pm to 2.0 pm, preferably 0.65 pm; and / or3-8) at least two hollow fiber filters are supported within the housing and are protruding with their open proximal ends through the closed end of the housing, with the hollow fiber filters preferably being arranged coaxially with each other and with the central longitudinal axis of the housing;4. The cell retention device according to any one of the preceding claims, wherein the cell retention device is for perfusion of mammalian cell suspension culture.

5. A microbioreactor (10) comprising a culture vessel (11) having a working volume of about 15 ml or less; a cell retention device (1) according to any one of the preceding claims; a mixing component (12) for mixing a content of the culture vessel; and an inlet port for introduction of culture medium to the culture vessel; wherein the cell retention device is arranged at least in part within the culture vessel, and wherein the microbioreactor is configured to allow removal of culture medium from the culture vessel through the at least one hollow fiber filter of the cell retention device, preferably in a continuous manner.

6. The microbioreactor according to claim 5, wherein one housing segment at the open end of the housing of the cell retention device protrudes further away from the closed end of the housing than another housing segment at the open end of the housing, the housing segments opposing each other, the open end of the housing thereby providing a window in the housing, for exposing the hollow fiber filter to the culture vessel; and the mixing component is arranged within the culture vessel opposite to the window, in regard to the housing of the cell retention device.

7. The microbioreactor according to claim 5 or 6, further comprising7-1) a source of negative pressure, preferably in the form of a pump, connected to the proximal end of the hollow fiber filter, for drawing culture medium out of the culture vessel through the hollow fiber filter;7-2) an inoculation port for inoculation with cells to be cultured;7-3) an addition port for addition of antifoam and / or base;7-4) a port for extraction of cultured cells and / or supernatant;7-5) a gassing port system for addition of O2, N2 and / or CO2;7-6) a means for detection of flow rate;7-7) an internal sensor for detection of dissolved oxygen within the culture vessel;7-8) an internal sensor for measurement of pH within the culture vessel; and / or7-9) a temperature regulating element for heating or cooling a content of the culture vessel.

8. The microbioreactor according to any one of claims 5 to 7, wherein8-1) the inlet port is provided in the housing of the hollow fiber filter of the cell retention device;8-2) the mixing component comprises a stirrer, preferably wherein the stirrer is arranged on a shaft protruding into the culture vessel, further preferably wherein the stirrer is a pitched-blade stirrer or a Rushton turbine; and / or8-3) at least a portion of the inner surface of the culture vessel is surface-modified to resist adherence of cells and / or proteins.

9. Use of a cell retention device according to any one of claims 1 to 4 for filtering culture medium out of a microbioreactor according to any one of claims 5 to 8.

10. Use of a microbioreactor according to any one of claims 5 to 8, for seed train culture, inoculum train culture or a main stage fermentation.

11. Method of exchanging culture medium in a microbioreactor according to any one of claims 5 to 8 by means of a cell retention device according to any one of claims 1 to 4, comprising the steps of: a) cultivating cells within the culture vessel by means of the culture medium; and b) removing culture medium through the at least one hollow fiber filter of the cell retention device; c) replacing removed culture medium with fresh medium through the inlet port of the microbioreactor or of the cell retention device, either continuously at a constant flow rate, or intermittently with a bolus injection of fresh culture medium.

12. The method according to claim 11 , wherein step b) is a continuous process; and / or wherein step c) is a continuous process with a constant flow rate of culture medium into the culture vessel; or wherein step c) is carried out intermittently with a bolus injection of fresh culture medium; and / or wherein steps a) to c) are repeated.

13. System of at least two microbioreactors according to any one of claims 5 to 8, wherein the system is configured to operate the microbioreactors in parallel.

14. The system according to claim 13, comprising12, 24, or 48 microbioreactors according to any one of claims 5 to 8; a holder configured to hold each microbioreactor in place during operation; a pumping system, preferably wherein the pumping system is at least in part pressure driven; at least one filling level sensor for a microbioreactor for monitoring the culture medium level in the microbioreactor; a fresh culture medium reservoir in fluid communication with each of the microbioreactors, for addition of fresh culture medium into each culture vessel; a waste reservoir in fluid communication with each of the microbioreactors for retention of culture medium removed from each of the microbioreactors; a heating / cooling system; a control unit configured to effect addition of fresh culture medium, optionally in response to a signal obtained from a filling level sensor, and / or to maintain a steady temperature across the system by controlling a heating / cooling system.

15. The system according to claim 13 or 14, wherein the system is configured to be controlled by a single operator.

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