Robust and efficient development and production system

Abstract

This invention relates to a system and methods for the cultivation or expansion of adherent cells, said system being a bioreactor system (1) comprising one or more impellers (3) directly connected with to a stirrer shaft (2), said impeller (3) comprising a basket (4) for holding a packed bed of carrier material; characterized in that said basket (4) comprises two or more chambers (21), wherein the walls of the chambers have a perforated surface (6), wherein said chambers are closed by a base plate (17), and optionally a lid (20); two, or more blades (5), wherein said blades are attached to said basket (4); and a bracket (18) at the center of at least one base plate (17) and / or of at least one lid (20), for connecting the basket (4) to the stirrer shaft (2).

Description

[0001] ROBUST AND EFFICIENT DEVELOPMENT AND PRODUCTION SYSTEM

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a system and methods for the cultivation or expansion of adherent cells, said system being a bioreactor system (1) comprising one or more impellers (3) directly connected with a stirrer shaft (2), said impeller (3) comprising a basket (4) for holding a packed bed of carrier material; characterized in that said basket (4) comprises two or more chambers (21), wherein the walls of the chambers have a perforated surface (6), wherein said chambers are closed by a base plate (17), and optionally a lid (20); two, or more blades (5), wherein said blades are attached to said basket (4); and a bracket (18) at the center of at least one base plate (17) and / or of at least one lid (20), for connecting the basket (4) to the stirrer shaft (2).

[0004] BACKGROUND OF THE INVENTION

[0005] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, viruses, vaccines, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require over 10 x 8 cells or of viruses, or over 10 x 11 extracellular vesicles (EVs). As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0006] There is a strong need for reliable and efficient platforms to scale up adherent cell culture. Traditionally, the culturing of adherent cells is performed using two- dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and HYPERStack® vessels. These approaches can have significant drawbacks, including the difficulty in achieving cellular density and cell numbers high enough to make it feasible for large scale production of therapies or cells.

[0007] Alternative methods have been suggested to increase volumetric density of cultured adherent cells. These include microcarrier-attached cultivation of cells performed in stirred tank bioreactors, in which the cells are subject to constant mechanical shear stress, resulting in a significant impact on viability, proliferation and culture performance. Variations of stirred bioreactors such as vertical wheel bioreactors lack key components of process controls needed for process development and manufacturing. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space. However, process control and scalability are limited.

[0008] Fixed-bed bioreactors have recently been developed from porous or meshed materials to overcome scalability issues e.g., in virus production. Another example of a high-density culture system for anchorage dependent cells is a packed-bed bioreactor system. In this type of bioreactor, a carrier material is used to provide a surface for the attachment of adherent cells. Medium is perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen needed for the cell growth. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed in US4833083A, EP0682697A1 , and US5510262A.

[0009] EP4071229A1 discloses a bioreactor system for cell culture comprising a cell culture module mounted inside a container between the top plate and the impeller, wherein said cell culture module allows the cultivation of adherent cells, and wherein the cell culture module is fixed to the head plate.

[0010] W02013043072A1 discloses a container for cultivating cell samples in a 3D matrix in a cell culture flask (“spinner flask”) using media containing serum. The container according to W02013043072A1 is specifically designed and explicitly intended for use in spinner flasks. Spinner flasks are cultivation vessels without process control for small scale use only, primarily for experimental purposes. In W02013043072A1 , the stirrer is a magnetic stirrer that is inserted into a specially designed magnetic holder. The container is not acting as stirrer.

[0011] US5705390A discloses a radial flow bioreactor comprising a reaction vessel and at least one basket within the reaction vessel having side wall perforations. The basket is filled with microcarriers and has a radial flow of medium through it. In this process, medium is pumped through a central inlet tube. Furthermore, a rotational movement of the basket, which is equipped with a ring of turbine blades, is described.

[0012] WO2017146928A1 discloses a perfusion bioreactor and a method for using the perfusion bioreactor to perform continuous cell culture. Mammalian cells are used to produce therapeutic proteins, monoclonal antibodies, viral vectors, and even cultured meat. Furthermore, in tissue engineering and regenerative medicine billions of stem cells are used to fabricate tissue engineered constructs or to replenish lost or damaged cells in degenerative diseases. Although suspension cell culture is widely used to produce proteins and antibodies, adherent cell culture is relevant to produce viral vectors for gene and modified cell therapy, as well as stem cells for regenerative medicine. Viral vectors are commonly used to deliver genetic materials into cells and tissues so that genetic defects can be corrected, cellular and tissue function be enhanced, or the production of cellular products be improved, ultimately leading to potential curative treatment.

[0013] Stem cells hold great promise for cell therapy, tissue engineering, and regenerative medicine as well as pharmaceutical and biotechnological applications. However, cells used for viral vector production are frequently anchorage-dependent; similarly, stem cells such as induced pluripotent stem cells (iPSCs) and, above all, mesenchymal stromal / stem cells (MSCs) are also inherently anchorage-dependent.

[0014] Mizukami et al. reported a yield of only 18 % of umbilical cord blood MSCs after expansion in a packed-bed bioreactor, which consisted of immobilized Fibra-Cel® disks in a disposable sterile bottle (Mizukami et al., 2013). dos Santos et al. disclosed the use of a xenogeneic free microcarrier suspension culture system to produce around 108bone marrow MSCs and 4.5 x 107adipose tissue derived stem / stromal cells (ASC) in a 1 L controlled stirred-tank bioreactor (dos Santos et al., 2014).

[0015] Wang et al. disclosed the continuous propagation of various anchorage dependent cells, including CHO, BHK, VERO, and hybridomas in a bioreactor equipped with a packed bed contained within a stationary packed-bed. The basket was suspended in the vessel of the bioreactor and the culture system was operated in perfusion mode. Using hybridoma cells, dos Santos et al. were able to reach high levels of monoclonal antibodies (mAb) in low serum levels or serum-free media (Wang et al., 1992).

[0016] Meuwly et al. disclosed that the theoretical maximum depth and diameter of a traditional, stationary, sandwiched packed bed is 0.03 m and 2 m, respectively. This maximal size is due to unavoidable gradients of nutrient concentrations linked to the depth of the packed bed and the ability to uniformly distribute the flow over the diameter of the packed bed (Meuwly et al., 2007).

[0017] There is a strong need for a robust, reliable, efficient, reproducible, and fully controlled bioreactor system to scale up adherent cell cultures, especially as more stem cell and EV-based therapeutic interventions enter the clinic. Moreover, maximizing cell yield needs to be safe and GMP-compliant in order to meet clinical standards. In some clinical applications large cell doses of more than 1 x 106mesenchymal stem / stromal cells (MSCs) per kg and 1 x 1010to 5 x 1011EVs per dose are needed.

[0018] SUMMARY OF THE INVENTION

[0019] The problem is solved by the embodiments of the present invention.

[0020] The systems and methods described herein provide a solution to the need for a robust, reliable, consistent, safe, and specifically GMP-compliant production setup and process for the cultivation and expansion of adherent cells and the use thereof to produce economically and medically important products with a high batch-to-batch consistency. Importantly, serum and human platelet lysate (hPL)-free media are used in this setup.

[0021] The present invention also provides a novel scalable agitated packed bed bioreactor system to overcome the hampered flow of nutrients inside traditional packed beds, thus surpass the theoretical size limitation of traditional packed bed bioreactor systems and hence improving cell growth and increasing cell densities as well as cell and product yields.

[0022] It has been shown by the inventors that seeding of cells in the case of an agitated packed bed according to the system and methods described herein resulted in a homogenous distribution of the cells on the carrier material. Moreover, it could be shown by the inventors that MSC and telomerized mesenchymal stem / stromal cells (MSC / TERT) grow well on two- and three-dimensional carrier material, specifically a more than 10-fold expansion could be shown for MSC / TERTs on a three-dimensional woven carrier material.

[0023] The present invention further provides a bioreactor system comprising a packed bed with a tunable attachment surface to culture volume ratio while also allowing flexible tuning of the media and media composition. Specifically, it has been shown by the inventors that cultivation of adherent cells using a scalable stirred bioreactor system with an agitated packed bed overcomes the drawbacks faced with a stirred bioreactor system using suspended microcarriers.

[0024] According to the invention, there is provided a bioreactor system (1) comprising one or more impellers (3) directly connected with to a stirrer shaft (2) for cultivation or expansion of adherent cells, said impeller (3) comprising a basket (4) for holding a packed bed of carrier material; characterized in that said basket (4) comprises two or more chambers (21), wherein the walls of the chambers (21) have a perforated surface (6), wherein said chambers (21 ) are closed by a base plate (17), and optionally a lid (20); two, or more blades (5), wherein said blades (5) are attached to said basket (4); and a bracket (18) at the center of at least one base plate (17) and / or of at least one lid (20), for connecting the basket (4) to the stirrer shaft (2).

[0025] According to a specific embodiment of the invention, the blades (5) are mounted on the outer surface of the basket (4).

[0026] According to a specific embodiment of the invention, the base plate (17) and / or lid (20) have a perforated surface (6).

[0027] According to a further embodiment of the invention, the bracket (18) comprises an elevated platform with a central hole (19), specifically said bracket (18) is a hollow cylinder.

[0028] According to a specific embodiment of the invention, the surface perforations (6) are circular, elliptical, triangular, quadrilateral, polygonal perforations, or a combination thereof, specifically of any shape geometries resulting from the union, intersection, difference, exclusion, or division of two or more shapes, or a combination thereof.

[0029] According to a specific embodiment of the invention, the surface perforations (6) range from 0.1-10 mm, specifically said surface perforations (6) have different diameters.

[0030] According to a specific embodiment of the invention, the blades (5) are mounted on the outer surface of the base plate (17), or the lid (20).

[0031] According to a further embodiment of the invention, the impellers (3) are made from a material selected from the group consisting of steel, stainless steel, aluminum, glass, ceramic, polyoxymethylene (POM), polyphenylene sulfone (PPSU), polypropylene (PP), polyether ether ketone (PEEK), ethylene-propylene-diene (EPDM), polyethylene (PE), polyamide (PA), silicone, printable resins, and bioplastics, specifically said impellers (3) are made of a corrosion resistant and sterilizable material.

[0032] According to a specific embodiment of the invention, the basket (4) has a volume of 0.01-50 L.

[0033] A further embodiment of the invention relates to a method for cultivating, or for expanding adherent cells using a bioreactor system (1) as described above.

[0034] According to a specific embodiment of the invention, the adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

[0035] According to a specific embodiment of the invention, the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, telomerized mesenchymal stem cells (MSC / TERT), HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs).

[0036] According to a specific embodiment of the invention, the cultivation or expansion of adherent cells is performed in batch, fed-batch, or perfusion mode.

[0037] According to a further embodiment of the invention, the pH, temperature, dissolved oxygen (DO) concentration, feed perfusion rate, and agitation rate are controlled.

[0038] According to a specific embodiment of the invention, the carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers, and hydrogels, and wherein said carrier material is made of a bio-compatible material that allows cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics (e.g. polylactic acid (PI_A), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides and hyaluronic acid (HA). A further embodiment of the invention relates to a method for cultivating or for expanding adherent cells in a bioreactor system (1 ) as described above, comprising the sequential steps of:

[0039] (a) preparing, equipping, and assembling the bioreactor system (1) as required, specifically filling said basket (4) with a carrier material, and mounting the one or more impellers (3) to the stirrer shaft (2);

[0040] (b) sterilizing said bioreactor system (1);

[0041] (c) adding a cultivation medium to the bioreactor system (1);

[0042] (d) seeding cells into the bioreactor system (1);

[0043] (e) agitating the cultivation medium with said stirrer shaft (2) under conditions to allow cells to adhere to the carrier material;

[0044] (f) exchanging the cultivation medium in perfusion mode and harvesting of conditioned medium;

[0045] (g) stopping the bioreactor system (1);

[0046] (h) dissociating the adherent cells from the carrier material; and

[0047] (i) collecting the dissociated cells.

[0048] According to a specific embodiment of the invention, the adherent cells of said method are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC) and immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs).

[0049] According to a specific embodiment of the invention, the carrier material of said method is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers and hydrogels, and wherein said carrier material is made of a bio-compatible material for cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PI_A), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

[0050] According to a specific embodiment of the invention, the adherent cells of said method are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

[0051] According to a specific embodiment of the invention, the adherent cells were seeded with a density of 1000-15000 cells / cm2.

[0052] According to a specific embodiment of the invention, the agitation is increased in a stepwise manner from 0 to 500 rpm, more specifically the agitation is increased stepwise in steps of 10-50 rpm with a final speed of 50-500 rpm, preferably the agitation has a final speed of 60-80 rpm.

[0053] A further embodiment of the invention relates to the use of the bioreactor system (1 ) and / or the methods as described above for producing viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells.

[0054] A further embodiment of the invention relates to a method for producing any one of viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells using the bioreactor system (1) and / or the methods outlined above.

[0055] FIGURES

[0056] Fig. 1 : Bioreactor system.

[0057] Fig. 2: DASbox bioreactor headplate setup.

[0058] Fig. 3: Basket, pictorial view 1.

[0059] Fig. 4: Basket, pictorial view 2.

[0060] Fig. 5: Basket, pictorial view 3.

[0061] Fig. 6: Basket, pictorial view 4.

[0062] Fig. 7: Basket, bottom view.

[0063] Fig. 8: Basket, top view.

[0064] Fig. 9: Basket, side view.

[0065] Fig. 10: Microscopic images of live and dead stained (Calcein-AM / DAPI) WJ- MSC / TERT273 cells adhered to Cytiva Cytodex 1 Microcarriers and Solohill Plastic Microcarriers. The images include transmitted light (trans) microscopy to visualize the cells and microcarriers, as well as fluorescence microscopy in the green fluorescent channel. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces.

[0066] Fig. 11 : Bioreactor process data of Solohill Plastic Microcarrier-attached WJ-

[0067] MSC / TERT273 in Mesencult ACF Plus Media. Fig. 12: Microscopic images of live and dead stained (Calcein-AM / DAPI) WJ- MSC / TERT273 attached to Cytiva Cytodex 1 Microcarrier in Mesencult media after 36 / 50 / 189 hours of dynamic attachment / cultivation in bioreactor (scale bar: 750 pm). To visualize cells and microcarriers, the images include fluorescence microscopy in the green fluorescent channel and overlays with transmitted light (trans) microscopy. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces.

[0068] Fig. 13: Microscopic images of live and dead stained (Calcein-AM / DAPI) WJ- MSC / TERT273 attached to Solohill Plastic Microcarriers MSC in MSC Nutristem XF (Sartorius) media supplemented with 5% HPL (PLT Gold, Sartorius) after 115 / 165 / 187 / 211 hours of dynamic attachment / cultivation in bioreactor (scale bar: 750 pm). To visualize cells and microcarriers, the images include fluorescence microscopy in the green fluorescent channel overlays with transmitted light (trans) microscopy and microcarriers. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces.

[0069] Fig. 14: Microscopic images of live and dead stained (Calcein-AM / DAPI) MSCs grown on Fibra-Cel® disks from different position inside the packed bed (scale bar: 750 pm). To visualize cells on Fibra-Cel® disks, the images include fluorescence microscopy in the green fluorescent channel. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces.

[0070] Fig. 15: Metabolic rates per day of WJ-MSC / TERT273 cultivated on Fibra-Cel® Disks over 750 hours.

[0071] Fig. 16: Flow Cytometry analysis of MSC associated surface proteins after bioreactor cultivation.

[0072] Fig. 17: Measured metabolites and particle concentrations of CP- MSC / TERT308 cultivated in a stirred packed bed over 550 hours.

[0073] Fig. 18: Microscopic images of cells on Fibra-Cel® discs after 3D differentiation stained for specific neural (Nestin and MAP2) and iPSC markers (TRA-1-60 and Oct3 / 4). The absence of Oct3 / 4 and the very low signal of TRA-1-60 confirm the differentiation of the iPSCs. Positive staining for Nestin and MAP2 verify the differentiation to the neural pathway. The microscopic pictures are an overlay of green (marker staining) and blue (DAPI, dead cell staining) channel images (scale bar: 750 pm).

[0074] Fig. 19: Mixing performance of a bioreactor system with an impeller comprising a basket filled with Fibra-Cel® disks at 80 rpm.

[0075] Fig. 20: Different views of an impeller.

[0076] DETAILED DESCRIPTION

[0077] Providing improved systems and methods to improve the yield and quality of cells, as well as the yield of cell-derived products, has strong economic and medical implications.

[0078] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person.

[0079] Culture of anchorage dependent cells has conventionally been performed by attaching the cells to stacked culture dishes or by growing the cells in roller bottles. These approaches have multiple drawbacks, such as increased batch-to-batch variability, low volumetric cell density, lack of adequate nutrient supply necessary to the growth of the cells, and limited scalability. In addition, cells tend to detach from their anchorage surface under conditions such as high or low serum concentrations, monolayer saturation or viscous sheer caused by stirring or perfusion of the culture medium.

[0080] The present invention relates to a stirred tank bioreactor system comprising one or more impellers comprising a basket which holds a bed of cell carriers, and which offers significant advantages for highly sensitive and anchorage-dependent cells such as MSCs. Such a system is able to generate homogenous conditions, even at high cell densities, over extended cultivation processes. By applying media perfusion and precisely controlled conditions, the system closely mimics physiological conditions and results in superior cell viability and product quality. Furthermore, such a system may be used for manufacturing purposes and can be scaled to different dimensions and is therefore favorable for process development towards manufacturing in GMP regulated environments.

[0081] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0082] The term “about” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0083] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0084] As used herein, “exemplary” or “for example” means “serving as an example, instance, or illustration”, and should not be construed as excluding other configurations, including those disclosed herein. As used herein, “such as” refers to (a) particular example(s) of the forgoing.

[0085] As used herein, processes conducted “in vitro” refer to processes which are performed outside of the normal biological environment.

[0086] As used herein, the terms “bioreactor system” and “bioreactor” are used interchangeably and refer to any device, fermentor, element, or system that supports a biologically active environment in which a biological process such as cultivation or expansion of cells under controlled conditions may be carried out. Bioreactors may be designed for small-scale cultures such as those used in research laboratories, as well as large-scale bioreactors comprising vessels or vats to produce and harvest biological macromolecules on a pilot plant or commercial scale. The bioreactor is a controlled environment wherein the oxygen / dO2 (dissolved oxygen), nitrogen, carbon dioxide, and pH levels may be adjusted.

[0087] The bioreactor system (1) described herein comprises a vessel (7) for holding a culture medium (displayed as 8 in Fig. 1), wherein said vessel (7) is made of, but not limited to, stainless steel, aluminum, glass, ceramic, polyoxymethylene (POM), polyphenylene sulfone (PPSU), polypropylene (PP), polyether ether ketone (PEEK), ethylene-propylene-diene (EPDM), polyethylene (PE), polyamide (PA), silicone, printable resins, polylactic acid (PI_A), or bioplastics, specifically said vessel (7) is made of a corrosion resistant and sterilizable material. Bioreactors may be “single-use” bioreactor based on a disposable material such as a disposable bag.

[0088] Specifically, the bioreactor system described herein is a “stirred tank bioreactor” (STBR) system. STBRs are scalable and can therefore be easily scaled up and be used on a large scale, as well as over a wide range of process parameters. STBRs are equably well suited for process development and production. Specifically, the bioreactor system described herein is not a spinner flask.

[0089] The bioreactor system (1) comprises a stirrer shaft (2), wherein said stirrer shaft (2) is agitated along its longitudinal axis, specifically said stirrer shaft (2) is rotated by a motor, specifically the shaft is rotated by a high precision motor with adjustable speed. Specifically, the stirrer shaft described herein is a fixed stirrer shaft.

[0090] The bioreactor system (1) can further comprise any one or more of a dissolved oxygen (DO) sensor (13), level sensor (16), temperature sensor (15), pH sensor (10), sampling and harvest port (9), seeding port (11), seeding port (11), feeding port (12), , submerged aeration port (14), headspace aeration port (23), an offgas (cooler) port (22), and a backup connection (24). Preferably, the bioreactor system (1) is of a “top stirrer” type.

[0091] The size of a bioreactor system (1) refers to the inner volume of the empty vessel (7) for holding a medium, specifically the size of the bioreactor system (1) may range from a few tens of milliliters to several cubic meters, specifically the size of bioreactors ranges from 0.05-1000 L, 0.05-750 L, 0.05-500 L, 0.05-250 L, 0.05- 100 L, 0.1-1000 L, 1-1000 L, 10-1000 L, 100-1000 L, 0.06-0.25 L, 0.1-0.25 L, 0.2- 1 L, 0.25-0.7 L, 0.32-1.25 L, 0.35-1 L, 0.35-1.5 L, 0.4-1.5 L, 0.4-1.8 L, or 1.25- 3.75 L, specifically 0.05, 0,075, 0.1 , 0.2, 0.25, 0.32, 0.35, 0.38, 0.4, 0.5, 0.75, 1 , 1.5, 1.8, 2, 5, 10, 12, 14, 15, 20, 25, 50, 75, 100, 250, 500, 750, or 1000 L.

[0092] The bioreactor system (1) may further comprise sensors selected from, but not limited to, metabolite sensors (e.g., glucose, lactate), Raman spectrometer, gas pressure sensor, dCO2 sensor, offgas sensors (e.g. N2, O2, CO2), volume level sensor, gas flow meter sensor, biomass sensor, optical sensors, electrochemical sensor, foam sensor, turbidity sensor, absorbance sensor, and scales for feed, harvest and bioreactor vessel.

[0093] In one aspect, the vessel (7) is made of a material selected from the group consisting of glass, plastic, and metal, preferably the vessel (7) is a glass vessel.

[0094] In one aspect, the inner wall of the vessel (7) is coated. Specifically, the inner wall of the vessel is coated with a siliconizing reagent for glass and other surfaces, specifically chlorinated organopolysiloxane in heptane, specifically Sigmacote®.

[0095] As used herein, the term “controlled parameters” refers to the physicochemical parameters that are monitored and controlled in real-time in a bioreactor system (1). Said parameters include, but are not limited to, pH, dissolved oxygen (DO), dissolved carbon dioxide (dCC ), glucose, lactate, temperature, volume, biomass, agitation speed, medium exchange rate, and feed or perfusion rate.

[0096] As used herein, preparing a bioreactor system (1) consists of choosing the appropriate type of bioreactor for the given application, checking for maintenance, making sure all bioreactor parts are complete and on hand, making sure to the all the parts of the bioreactor system (1) are clean, making sure all mechanical parts are working properly, and making sure all parts are undamaged.

[0097] As used herein, “equipping” a bioreactor system (1) consists of selecting the appropriate sensors, selecting the appropriate ports and connections, and selecting appropriate additional material such as material, for sampling and perfusion of the bioreactor.

[0098] As used herein, “assembling” a bioreactor system (1) consists of assembling said bioreactor according to the manufacturers’ specifications and as required for the relevant bioreactor process.

[0099] As used herein, “stopping” a bioreactor system (1) consists of harvesting the products of interest (e.g., cells, supernatant), turning of the electronics according to the manufacturers’ specifications, decontaminating said bioreactor system (1) according to an appropriate protocol, and cleaning said bioreactor system (1).

[0100] As used herein, the term “impeller” (3) refers to a device connected to, specifically directly connected to or directly mounted on, a stirrer shaft (2) of a bioreactor system (1) and arranged to move and mix the medium in said bioreactor system (1) when rotated. Said impeller (3) may be made from any material selected from steel, stainless steel, aluminum, glass, ceramic, polyoxymethylene (POM), polyphenylene sulfone (PPSU), polypropylene (PP), polyether ether ketone (PEEK), ethylene-propylene-diene (EPDM), polyethylene (PE), polyamide (PA), silicone, printable resins, polylactic acid (PI_A), and bioplastics, specifically said impellers (3) are made of a corrosion resistant and sterilizable material. One non-limiting embodiment of an impeller is shown in Fig. 20.

[0101] As used herein, the term “agitation” refers to the resulting mixing effect of an impeller (3) connected to a stirrer shaft (2) which is rotating, i.e., circular movement, along its longitudinal axis.

[0102] As used herein, the “agitation speed”, or synonymously the rotational speed or agitation rate, can range from 1-2000 rpm, 25-1500 rpm, 25-1200 rpm, 5-500 rpm, 5-250 rpm, 5-200 rpm, 5-150 rpm, or 50-500 rpm, specifically the agitation speed is 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 rpm. The agitation speed can be constant or variable, specifically the desired agitation speed can be reached in a stepwise manner, wherein the step size is 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 500, or 1000 rpm, and wherein the length of the steps can range from 1-360 seconds, specifically the length of the steps is 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210, 240, 300, or 360 seconds. In a preferred embodiment the final agitation speed is in the range of 60-80 rpm, specifically 50-80 rpm, and is reached in steps of 10-50 rpm.

[0103] As used herein, the term “bracket” (18) refers to a specific part on the outer surface of an impeller (3) for connecting said impeller (3) to a stirrer shaft (2), specifically the bracket (18) refers to a set-screw collar on the outer surface of an impeller (3). There may be one or two brackets (18) on one impeller (3).

[0104] As used herein, the term “directly connected” with respect to a basket and a stirrer shaft is meant to describe that the basket is directly attached to a stirrer shaft to join or link the basket and the stirrer shaft together. In this context, the terms connected, attached, coupled, linked, joined, and bound are to be understood as synonyms.

[0105] As used herein, the term “basket” (4) refers to a container element with a perforated surface comprising one or more chambers for holding a packed bed of carrier material, wherein said chambers are capped on the nadir end by a cover referred to as “base plate” (17) and may, on the opposite zenith end, be capped with a cover referred to as “lid” (20). The volume of said basket (4) ranges from 0.01-100 L, 0.01-100 L, 0.01-50 L, or 0.1-100 L, specifically 0.01 , 0.025, 0.05, 0.075, 0.1 , 0.15, 0.2, 0.25, 0.5, 0.75, 0.8, 1 , 1.2, 1.5, 1.8, 2, 2.5, 5, 10, 12, 15, 20, 25, 50, 75, or 100 L. The “surface perforations” (6) of said basket (4) may be circular, elliptical, triangular, quadrilateral, polygonal perforations, or a combination thereof, specifically of any shape geometries resulting from the union, intersection, difference, exclusion, or division of two or more shapes, or a combination thereof. In a preferred embodiment, the basket comprises a central gap (25) or channel through which liquid may flow inside the basket. One embodiment of a basket is shown in Fig. 20. As used herein, impeller (3) “blades” (5) can be attached to the outer surface of a basket (4), specifically the blades (5) can be attached to the base plate (17) and / or lid (20) of said basket (4). In a preferred embodiment the blades (5) are equidistantly spaced rectangular plates. Specifically, the attachment of the blades (5) on the base plate (17) and the lid (20) enables efficient mixing of the culture medium within and between the upper and lower part of the vessel.

[0106] As used herein, a “packed bed” refers to a hollow container filled with a plurality of matrices having been packed together in at least a portion of a hollow container, wherein said hollow container refers to a basket (4), specifically to a chamber (21) of said basket (4).

[0107] As used herein, “agitated” or “rotating”, when directed to a packed bed, refer to a packed bed that is agitated implicitly while located in a basket (4) connected to the stirred shaft (2).

[0108] As used herein, the term “holding”, when directed to a basket and a packed bed, refers to the ability of said basket to retain a packed bed.

[0109] As used herein, the term “carrier material” or “microcarrier” refers to a support matrix that allows for the growth of adherent cells. Said carrier material may be two- or three-dimensional, specifically said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers, and hydrogels. Specifically, said carrier material is made of a bio-compatible material that allows cell attachment of adherent cells, specifically selected from polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics (e.g. polylactic acid (PI_A), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, or hyaluronic acid (HA). Said carrier material may comprise surface modifications, specifically the surface modifications are selected from, but are not limited to, the group consisting of extracellular matrix proteins, functional groups, recombinant proteins, peptides, and charged molecules. In a preferred embodiment, the carrier material is loosely filled into the basket, specifically the basket is filled with 5-5.5 g carrier material, specifically the packed bed is filled with 6000-6600 cm2carrier material.

[0110] As used herein, “cultivation”, “culturing”, “cultivating”, or “growing”, when directed to a cell or cells, refer to a method step to propagate, expand or maintain a population of cells in culture media of various kind. Conventional methods and techniques are well-known to the skilled person in the field of molecular biology, biology, biochemistry, genomics, cell culturing and the like. Although the term “cultivation” is generally understood to include the proliferation or division of cells, it also includes methods of differentiating cells in culture medium. Proliferation and differentiation are not mutually exclusive and may happen at the same time in the same culture medium.

[0111] As used herein, “expanding” or “expansion” refers to culturing one or more cells for the purpose of obtaining a larger number of cells in the culture.

[0112] The terms “culture media”, or “medium” also, and preferably, include media that are suitable for the in vitro cell culture of human or animal cells for a prolonged period of time. Such culture media comprises sufficient components to allow the cells to grow, proliferate and / or differentiate over longer period of, for example, at least a day. A “defined culture media” refers to a (growth) medium suitable for the in vitro cell culture of human or animal cells and in which all of the chemical components are known. Such defined media does not or essentially not comprise any ill-defined source of nutrients and / or other ill-defined factors. As used herein, “culture media” or “media” refers to a liquid used to culture the host cells in the bioreactor. The media used in the method disclosed herein may include various ingredients that support the growth of the host cells, including but not limited to amino acids, vitamins, organic and inorganic salts, carbohydrates. The media may be serum-free media, which is media formulated without any animal serum. A serum-free media when used may be selected from among DMEM, DMEM / F12, Medium 199, MEM, RPMI, OptiPRO SFM, VP-SFM, VP- SFM AGT, HyQ PF-Vero, MP-Vero, MesenCult™-ACF Plus (Stem Cell Technologies), MSC Nutristem XF (Sartorius), StemScale™ PSC Suspension Medium (Gibco), or others. The culture media may also be animal-free media; that is, it does not have any product of animal origin. The culture media may also be protein-free media; that is, the media is formulated with no proteins. The serum-free or protein-free media may be formulated without serum or protein but may contain cellular protein derived from the host cells, and optionally proteins specifically added to the serum-free or the protein- free media. The culture media may also be xenogeneic-free (xeno-free). Further examples of culture medium are, serum- and human platelet lysate (hPL)-free cultivation media, and chemically-defined media. The pH for cultivation can be, for example, between 6.5-7.5, depending on the pH stability of the host cells. Preferably the cells are cultivated at a pH of 7.2-7.4. The host cells may be cultivated at the temperature between 20-40 °C., specifically between 30° C. and 40° C., and preferably at 36° C.±1 ° C. for mammalian cells.

[0113] As used herein, the term “conditioned”, when directed to culture medium, refers to a culture medium in which cells have been or are grown, specifically a conditioned medium contains cell products such as secreted cell products, extracellular vesicles, proteins, antibodies, lipids, or waste and cell debris. Conditioned medium may also be referred to as cell culture supernatant, spent medium, or depleted medium.

[0114] As used herein, the term “seeding”, when directed to cells, refers to the inoculation of cells into a bioreactor system (1 ). Specifically, the number of cells seeded into a bioreactor system (1) is determined beforehand. In a preferred embodiment, 100-200000 cells / cm2specifically 1000-150000 cells / cm2, more specifically 2000- 150000 cell / cm2, specifically 2000-100000 cell / cm2, specifically 2000-50000 cell / cm2, specifically 2000-20000 cell / cm2even more specifically 2000-15000 cell / cm2carrier material are seeded into a bioreactor system (1). A seed density of 2000 cells / cm2corresponds to approx. 5.3 x 104cells / mL in the bioreactor system described herein.

[0115] As used herein, “agglomerates”, “aggregate”, “aggregation”, and “aggregated” in connection to cells refer to one of several main types of cell organization, namely the joining or clustering of a cell with another cell, or cells. Moreover, it does not comprise the joining of a cell with a substrate, commonly referred to as “adherence”. Aggregation of cells is based on cell-cell interactions. Such interactions can be formed between cells through cell surface proteins and are normally present in many biological systems such as tissues, organs and the like. Cell aggregation, when compared to a single cell, enhances survival and functioning of the cell.

[0116] As used herein, “adherent” or “anchorage dependent”, when directed to a cell or cells, refers to cells that require to be attached to a surface in culture condition in order to grow. Once the cells with a preference for adhesion are seeded into a bioreactor system (1) equipped with a packed bed filled with appropriate carrier material, said cells readily attach to said carrier material and on which they can proliferate. The cells adhered to the carrier material can be dissociated from said carrier material with methods such as e.g., sonication or through enzymatic dissociation or a combination of enzymatic and physical treatment. The preferred adherent cell is an anchorage-dependent cell that may be grown on a suitable carrier, but suspension cells that may be adapted to grow as adherent cells may also be used. The bioreactor system (1) may be used to propagate suspended and adherent cells. For example, adherent cells may be derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, or dental pulp. Specific examples of adherent cells include, but are not limited to, mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs), MBCK cells, MDBK cells, MRC- 5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, HeLa cells, HEK 293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, WI-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, and QOR cells. It is within the knowledge of one skilled in the art to select an adherent host cell suitable for use in the process of the invention.

[0117] Telomerization stabilizes MSCs (MSC / TERT) over many generations regarding genetic stability, the growth rate, and phenotype. Ectopic expression of human telomerase is safe and does not change MSC phenotype, marker expression, and biological functions. Specifically, WJ-MSC / TERT273 and other MSC / TERT lines display stable phenotype, marker expression, and growth properties over more than 90 population doublings. The MSC / TERT RNA and protein profiles are consistent between different batches. MSCs and MSC / TERTs act in a paracrine rather than a cellular manner.

[0118] Dissociated adherent cells are collected using methods from the group consisting of, but not limited to, centrifugation, filtration and tangential flow filtration (TFF).

[0119] The bioreactor system (1) and methods described herein can be used for production of biological products, specifically for producing viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), enucleated cells, proteins, antibodies, and lipids.

[0120] Specifically, said viruses which can be produced by the inventive methods and bioreactor system can be adeno-associated viruses (AAV), lentiviruses (LV) or others.

[0121] Extracellular vesicles (EVs) are particles that are released from cells and are delimited by a lipid bilayer, and are not able to replicate on their own. Specifically, also extracellular vesicles (EVs) can be produced. EVs are cell-derived membrane- surrounded vesicles that carry various types of bioactive molecules and deliver them to recipient cells. EVs can cargo pathophysiological biomarkers. Classical EVs are exosomes, microvesicles, and apoptotic bodies, autophagic EVs, matrix vesicles, and stress-induced EVs. The bioreactor system (1) and methods of the present invention may be used to cultivate cells to produce therapeutic EVs for use in regenerative medicine and / or the treatment of, but not limited to, stroke, osteoarthritis, acute lung inflammation (ALI), acute kidney injury, rheumatoid arthritis, or graft-versus-host- disease. Specifically, the bioreactor systems (1) and methods disclosed in the present invention may be used to produce target-specific EVs, specifically recombinant EVs with antibody-like affinity, to target cells and tissues (W02020035532A1). EVs isolated from mesenchymal stem / stromal cell lines have a lipid membrane, characteristic protein composition, and stable miRNA profiles over time and / or between different batches. Specifically, EVs that are produced by MSCs derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples have a biological activity such as an anti-inflammatory or an anti-fibrotic activity. No loss in activity is to be expected upon long-term storage of EVs, specifically storage of said EVs at -80 °C. . Specifically, the production of EVs in a 250 mL agitated packed bed bioreactor system can reach EV quantities of (5-15) x 103particles or more per cell per day, while reaching particle concentrations of up to 1 x 109particles per mL, preferably, 5 x 109particles per mL or more.

[0122] As used herein, the terms “nanovesicles” and “artificial cell-derived vesicles” (ACDVs) can be used interchangeably. Artificial cell-derived vesicles (ACDVs) are EV mimetics that are produced in the laboratory under conditions of induced cell disruption, such as extrusion. Specifically, starting material for cell-derived nanovesicles can be produced by the inventive system and methods. Nanovesicles are versatile systems for the targeted delivery and / or targeting of drugs, biomolecules, and contrast agents.

[0123] Specifically, starting material for enucleated cells can also be produced herein. Enucleated cells neither proliferate nor permanently engraft in the host, yet retain the organelles for energy and protein production, undergo integrin-regulated adhesion to inflamed endothelial cells, and actively home to chemokine gradients established by diseased tissues such as mesenchymal stromal cells with their nuclei removed by e.g. density gradient centrifugation.

[0124] “Perfusion mode”, when directed to cell culture, is well known in the art and refers to a culturing method for a bioreactor system (1) in which the cells are retained in the bioreactor (1) and the culture medium is exchanged, specifically the conditioned culture medium is harvested and replaced with fresh culture medium. Fresh medium is provided at the same rate that the conditioned medium is removed. The harvested culture medium does not contain cells. A cell culture in perfusion mode may be active for several months, specifically a cell culture in perfusion mode is stopped after 1- 365 days, 1-184 days, 1-62 days, 1-31 days, 1-21 days, 1-14 days, 1-7 days, or 1- 5 days, specifically a cell culture in perfusion mode is stopped after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , , 180, 200, 250, 300, or 365 days. In a preferred aspect, a cell culture in perfusion mode is stopped after 25-50 days, specifically 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 days, more specifically 28, 29, 30, or 49 days.

[0125] As used herein, the terms “feed rate” or “perfusion rate” refer to the rate at which the conditioned culture medium is exchanged with fresh medium.

[0126] “Batch mode”, when directed to cell culture, is well known in the art and refers to a culturing method for a bioreactor system (1) in which a substrate (or feed) is administered to a culture at the start of the bioprocess. The feed is consumed by the cells in said culture and by-products accumulate over time. Said culture is harvested once there is no substrate left to consume and the cells entered the stationary phase. A cell culture in batch mode may be repeated several times, specifically a cell culture in batch mode may be repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0127] “Fed batch mode”, when directed to cell culture, is well known in the art and refers to a culturing method for a bioreactor system (1) in which a culture is incrementally supplied with substrate over a certain period of time. This allows to oppose to a decline in cell growth or cell densities, observed in a batch mode culture method. A cell culture in fed batch mode may be active for several days, specifically a cell culture in fed batch mode is stopped after 1-14 days, 1-7 days, or 1-5 days, specifically a cell culture in fed batch mode is stopped after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days. In one aspect, the bioreactor system (1) as described herein is assembled as follows. The basket (4) of the impeller (3) is filled with carrier material, specifically a three-dimensional carrier material, to form a packed bed, and said impeller (3) is mounted, specifically connected, to the stirrer shaft (2). The bioreactor system (1) is assembled with offgas cooler, gas filters, ports, and tubes, tubings, sealing, and sensors. The pH (10) and DO (13) sensor are installed above the packed bed. The pH sensor (10) is calibrated using a pH range before sterilization of the bioreactor system (1). Specifically, the pH sensor is calibrated using pH 4 and pH 7 buffers before sterilization of the bioreactor system (1). Feed and harvest pumps are calibrated, specifically with the feed / harvest lines and pump head tubings. A buffer, specifically PBS (particle free), is added before sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing are prepared as required and sterilized as well. The DO sensor is calibrated using headspace aeration with 100 and 0 % oxygen, with the buffer-filled bioreactor after sterilization. In one aspect, sterilization is performed by autoclaving.

[0128] In one aspect, cultivation using the bioreactor system (1) as described herein is performed as follows. After sterilization, the buffer is removed, and cultivation medium is added to the bioreactor system (1). In one aspect, the cultivation medium is selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media. In one aspect, the culture medium is equilibrated to process conditions using automated process control, specifically the conditions are pH 7.2, DO 10 %, and 37 °C. Cells are seeded into the bioreactor system (1), the bioreactor is filled with cultivation media and agitation is started. In one aspect, cells are seeded into the bioreactor with a density of approximately ranging from 2000 to 3000 cells / cm2. In one aspect, the cells are adherent cells, specifically the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent stem cells (iPSCs). In one aspect, the cells are selected from the group consisting of MSC / TERT cell lines derived from placenta, umbilical cord, bone marrow, dental pulp or adipose tissue. In one aspect, cultivation media is exchanged in perfusion mode, specifically using two pumps and a level sensor to keep the volume constant. In one aspect, the dilution rate is controlled using off-line metabolite measurements.

[0129] The bioreactor system described herein allows a 2- to 30-fold expansion of cells, specifically 2- to 20-fold, more specifically 5- to 18-fold. Specifically, the bioreactor system described herein allows a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11 -, 12-, 13-, 14-, 15- , 16-, 17-, 18-, 19-, 20-, 21 -, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, or 30-fold expansion of cells. In a preferred aspect, the bioreactor system described herein allows a 5- to 18-fold expansion of cells. Specifically, the bioreactor system described herein allows the expansion of cells to final cell densities ranging from 1 x 104to 4 x 104cells per cm2, specifically of up to 1.1 x 1Q4, 1.5 x 104, 1.7 x 1Q4, 2 x 1Q4, 2.9 x 104, 3.2 x 104, 3.3 x 104, 3.5 x 104, or 3.8 x 104cells per cm2. Specifically, the bioreactor system described herein allows the expansion of cells to final cell densities ranging from 2.5 x 105to 106cells per mL, specifically of up to 2.9 x 105, 4 x 105, 4.4 x 105, 5.2 x 1 o5, 7.7 x 1 o5, 8.5 x 105, 8.7 x 1Q5, 9.1 x 1Q5, 1 x 1Q6cells per mL. Specifically, the bioreactor system described herein allows the harvest of extracellular vesicles ranging from 1 x 109to 1 x 1O10particles per mL, specifically 1 x 109to 5.5 x 109particles per mL, specifically up to 2 x 109, 2.7 x 109, 3.2 x 109, 3.5 x 109, 5.2 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, or 1 x 1O10particles per mL.

[0130] The present invention also encompasses the following embodiments:

[0131] 1. A bioreactor system (1) for cultivation or expansion of adherent cells comprising one or more impellers (3) coupled to a stirrer shaft (2), said impeller (3) comprising a basket (4) for holding a packed bed of carrier material; characterized in that said basket (4) comprises

[0132] (a) two or more chambers (21 ), wherein the walls of the chambers (21 ) have a perforated surface (6), wherein said chambers (21) are closed by a base plate (17), and optionally a lid (20);

[0133] (b) two, or more blades (5), wherein said blades (5) are attached to said basket (4);

[0134] (c) a bracket (18) at the center of at least one base plate (17) and / or of at least one lid (20), for connecting the basket (4) to the stirrer shaft (2).

[0135] 2. The bioreactor system (1) of embodiment 1 , wherein the blades (5) are mounted on the outer surface of the basket (4). 3. The bioreactor system (1) of any one of embodiments 1 or 2, wherein said base plate (17) and / or lid (20) have a perforated surface (6).

[0136] 4. The bioreactor system (1) of any one of embodiments 1 to 3, wherein said bracket (18) comprises an elevated platform with a central hole (19), specifically said bracket (18) is a hollow cylinder.

[0137] 5. The bioreactor system (1 ) of any one of embodiments 1 to 4, wherein the surface perforations (6) are circular, elliptical, triangular, quadrilateral, polygonal perforations, or a combination thereof, specifically of any shape geometries resulting from the union, intersection, difference, exclusion, or division of two or more shapes, or a combination thereof.

[0138] 6. The bioreactor system (1 ) of any one of embodiments 1 to 5, wherein the diameter of said surface perforations (6) ranges from 0.1-10 mm, specifically said surface perforations (6) have different diameters.

[0139] 7. The bioreactor system (1 ) of any one of embodiments 1 to 6, wherein the blades (5) are mounted on the outer surface of the base plate (17), or the lid (20).

[0140] 8. The bioreactor system (1) of any one of embodiments 1 to 7, wherein said impellers (3) are made from a material selected from the group consisting of steel, stainless steel, aluminum, glass, ceramic, polyoxymethylene (POM), polyphenylene sulfone (PPSU), polypropylene (PP), polyether ether ketone (PEEK), ethylene- propylene-diene (EPDM), polyethylene (PE), polyamide (PA), silicone, printable resins, and bioplastics, specifically said impellers (3) are made of a corrosion resistant and sterilizable material.

[0141] 9. The bioreactor system (1) of any one of embodiments 1 to 8, wherein said basket (4) has a volume of 0.01-50 L.

[0142] 10. A method for cultivating, or for expanding adherent cells using a bioreactor system (1 ) of any one of embodiments 1 to 9.

[0143] 11 . The method of embodiment 10, wherein said adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

[0144] 12. The method of embodiments 10 or 11 , wherein the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs).

[0145] 13. The method of any one of embodiments 10 to 12, wherein the cultivation or expansion of adherent cells is performed in batch, fed-batch, or perfusion mode.

[0146] 14. The method of any one of embodiments 10 to 13, wherein the pH, temperature, dissolved oxygen (DO) concentration, feed or perfusion rate and agitation rate are controlled.

[0147] 15. The method of any one of embodiments 10 to 14, wherein said carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers, and hydrogels, and wherein said carrier material is made of a bio-compatible material that allows cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PLA), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

[0148] 16. A method for cultivating or for expanding adherent cells in a bioreactor system (1) of any one of embodiments 1 to 9, comprising the sequential steps of:

[0149] (a) preparing, equipping, and assembling the bioreactor system (1) as required, specifically filling said basket (4) with a carrier material, and mounting the one or more impellers (3) to the stirrer shaft (2);

[0150] (b) sterilizing said bioreactor system (1);

[0151] (c) adding a cultivation medium to the bioreactor system (1);

[0152] (d) seeding cells into the bioreactor system (1);

[0153] (e) agitating the cultivation medium with said stirrer shaft (2) under conditions to allow cells to adhere to the carrier material;

[0154] (f) exchanging the cultivation medium in perfusion mode and harvesting of conditioned medium;

[0155] (g) stopping the bioreactor system (1);

[0156] (h) dissociating the adherent cells from the carrier material; and

[0157] (i) collecting the dissociated cells. 17. The method of embodiment 16, wherein the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC) and immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, induced pluripotent cells (iPSCs).

[0158] 18. The method of embodiment 16 or 17, wherein the carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers and hydrogels, and wherein said carrier material is made of a bio-compatible material for cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PLA), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

[0159] 19. The method of any one of embodiments 16 to 18, wherein said adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

[0160] 20. The method of any one of embodiments 16 to 19, wherein the adherent cells are seeded with a density of 1000-15000 cells / cm2carrier material.

[0161] 21. The method of any one of embodiments 16 to 19, wherein the adherent cells are seeded with a density of 1000-200000 cells / cm2carrier material.

[0162] 22. The method of any one of embodiments 16 to 21 , wherein the agitation is increased in a stepwise manner from 0 to 500 rpm, more specifically the agitation is increased stepwise in steps of 10-50 rpm with a final speed of 50-500 rpm.

[0163] 23. Use of the bioreactor system (1 ) of any one of embodiments 1 to 9 or use of the methods of any one of embodiments 10 to 22 for producing viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells.

[0164] 24. A method for producing any one of viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells using the bioreactor system (1 ) of any one of embodiments 1 to 9 or using the methods of any one of embodiments 10 to 22. The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the invention.

[0165] EXAMPLES

[0166] Example 1 : Cultivation of telomerized mesenchymal stem / stromal cells (MSC / TERTs) in a stirred tank bioreactor svstem using an impeller comprising a basket holding a packed bed composed of Fibra-Cel® Disks

[0167] Cell line and seed generation

[0168] The seed culture was prepared from 2D-expanded cells. A vial of 3 * 105- 5 * 105cells was thawed and seeded in 2D and grown at 37 °C and 5 % CO2 in an incubator. Cells were passaged at 80-90 % confluency every 3 to 4 days. For bioreactor seeding, cells were detached from culture flasks, counted, pelleted and resuspended in cultivation media, and then directly seeded into the bioreactor.

[0169] Cells were cultured in pre-coated cell culture flasks using commercial MSC cultivation medium (Attachment Substrate and Mesencult ACF Plus from Stem Cell Technologies or NutriCoat Attachment Solution and MSC Nutristem XF from Sartorius) at 37 °C and 5 % CO2.

[0170] The applied MSC lines were stabilized by telomerization using a fragment of human telomerase (hTERT) (Wolbank et al., 2009).

[0171] Bioreactor system preparation and setup

[0172] A DASbox bioreactor (Eppendorf) was used for the experiment. Glass vessel coating (Sigmacote, Sigma-Aldrich) was performed to prevent cell attachment to said vessel. The basket of the impeller was filled with carrier material (5-5.5 g Fibra-Cel® disks) to form a packed bed, and said impeller was mounted to the stirrer shaft. The bioreactor system was assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (see Fig. 2 for exemplary bioreactor headplate setup). The pH and DO sensor are installed above the packed bed using a PG 13.5 port (ID 12 mm). The pH sensor was calibrated using pH 4 and pH 7 buffers before autoclaving. Feed and harvest pumps were calibrated with the feed / harvest lines and pump head tubings. PBS (particle free) was added before autoclaving for sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing were prepared as required and sterilized as well. The DO sensor was calibrated using 100 and 0 % oxygen, with the PBS-filled bioreactor after autoclaving.

[0173] Bioreactor system cultivation

[0174] PBS was removed, and cultivation medium (Mesencult ACF Plus from Stem Cell Technologies or MSC Nutristem XF from Sartorius) was added to the bioreactor system and equilibrated to process conditions using automated process control (pH 7.2, DO 10 %, 37 °C). Cells (e.g., MSC / TERT cell lines derived from placenta, umbilical cord, bone marrow, dental pulp or adipose tissue) were seeded with a density of approximately 2000 cells / cm2into the bioreactor system and agitation was started. The agitation rate was started with 30-50 rpm, and slowly increased to 60-80 rpm. Cultivation media was exchanged in perfusion mode, using two pumps and a level sensor to keep the volume constant. The dilution rate was controlled using off-line metabolite measurements.

[0175] Sampling and evaluation

[0176] Samples were taken from the cultivation, for metabolite analysis, particle measurement, and microscopic imaging of the supernatant. Staining with Calcein-AM and DAPI was used to visualize live and dead cells.

[0177] Analysis of the final harvest

[0178] Disks of different parts of the packed bed were analyzed to verify homogenous conditions. Cells were stained with Calcein-AM and DAPI to check viability. CellTiter- Glo 3D assay was performed to determine the amount of viable cells per disk. Some disks were frozen for miRNA analysis.

[0179] The rest of the disks were used for dissociation of cells which was started by Versene treatment of disks after a PBS wash. Afterwards an enzyme cocktail (5 mM EDTA solution, Accutase and TrypLE Select) was applied to the disks. In order to free the cells from the fibrous mesh inside the disks, they were sonicated using an ultrasound waterbath. Detached cells were counted and used for flowcytometry analysis of MSC markers.

[0180] Results

[0181] Five telomerized MSC lines (i.e., WJ-MSC / TERT273, CP-MSC / TERT308, P-MSC / TERT308, BM-MSC / TERT292, and ASC / TERT300) were successfully cultivated in a bioreactor system as described herein for up to 49 days (Table 1). Cell viability, metabolic activity, and productivity of extracellular vesicles (particles) remained high throughout the whole process.

[0182] Table 1 : Performance of telomerized Cell Lines in stirred tank bioreactor system using an impeller comprising a basket holding a packed bed composed of Fibra-Cel® Disks.

[0183] Fig. 14 shows microscopic images of live and dead stained (Calcein-AM / DAPI) MSCs grown on Fibra-Cel® disks from different positions inside the packed bed (scale bar: 750 pm). To visualize cells on Fibra-Cel® disks, the images include fluorescence microscopy in the green fluorescent channel. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces. The cells were well attached and highly viable. The upper 3 pictures in Fig. 14 show disks from the top of the packed bed. The middle row shows disks from the middle and the lower ones show disks from the bottom of the packed bed after 23 days of cultivation when the reactor was opened.

[0184] Fig. 15 shows metabolic rates per day of WJ-MSC / TERT273 cultivated on Fibra-Cel® Disks over 750 hours. Glucose consumption rate is increasing because cells were growing in the bioreactor. Lactate production rate is in the same range as the glucose consumption rate because of 1 :1 conversion. Ammonia production rate is increasing due to higher Glutamine consumption of growing cells.

[0185] Cells were found to attach well to Fibra-Cel® Disks during the attachment phase (observed in small scale experiments) and were observed to grow to high confluency over the cultivation process (Fig. 14). Highly viable and metabolically active cells were observed for up to 32 days (Fig. 15). The multipotent state of the MSC was confirmed by Flow Cytometry analyzing two established negative markers (CD34 and CD45) and four established positive MSC markers (CD44, CD73, CD90 and CD105). Cells showed a downregulation of CD105 after cultivation on Fibra-Cel® Disks, which has been associated with increased immunomodulatory capabilities of MSCs (Antebi et aL, 2018; Pham et aL, 2019) (Fig. 16).

[0186] Fig. 16 shows a flow Cytometry analysis of MSC associated surface proteins after bioreactor cultivation. CP-MSC / TERT308 cells cultivated on Fibra-Cel® Disks over 550 hours were detached from the disks and incubated with different antibodies targeting extracellular proteins. Each antibody staining (Fig. 16) was overlayed with the corresponding isotype control staining. MSCs cultivated on Fibra-Cel® Disks do not express the negative markers CD34 or CD45 on their surface whereas they show high expression of the positive markers CD44, CD73 and CD90. A decreased expression was detected for the positive marker CD105.

[0187] The final harvest of a 23-day perfusion process with placenta-derived MSCs (telomerized) resulted in 2.2 x 108total cells (CellTiter-Glo 3D Assay), which corresponds to 868.000 cells / mL or 33.000 cells / cm2(Table 2). Disks from different positions of the packed bed were shown to be similarly occupied by cells (Fig. 14).

[0188] Fig. 17 shows the measured metabolites and particle concentrations of CP- MSC / TERT308 cultivated in a stirred packed bed over 550 hours. The perfusion rate was adapted to the glucose concentration in the medium to keep the glucose concentration at a constant level. Particle concentrations are in a range of 1.5 x 109- 2.7 x io9particles per mL of supernatant. The particle concentration is decreasing towards the end of the process due to a strongly increased perfusion rate.

[0189] The process produced 6.8 L harvest with a particle concentration of 1.3 x 109- 2.6 x 109particles / mL (NTA), which corresponds to 1.3 x 1013total particles (Fig. 17).

[0190] In another process using the bioreactor system as described herein with umbilical cord-derived MSCs, that was performed with batch-wise media exchange for 32 days, particle production was in a similar range (109particles / mL).

[0191] Disks from different regions of the packed bed (top, middle and bottom) were subjected to viability analysis. The luminescence signal generated from each disk was compared to a standard curve generated with known cell numbers and an overall mean of the bioreactor was calculated. Table 2: CellTiter-Glo 3D analysis of CP-MSC / TERT308 grown Fibra-Cel® Disks over 550 hours.

[0192] Cells / disk Total cells BRC Cells / ml Cells / cm2

[0193] Upper 2.3 x io52.5 x 10810.0 x 1053.8 x 104

[0194] Middle 1.9 x 1O52.1 x 1088.5 x 1053.2 x 104

[0195] Lower 1.7 x 1051.9 x 1087.6 x 1052.9 x 104

[0196] Overall mean 2.0 x 1O52.2 x 1088.7 x 1053.3 x 104

[0197] Example 2: Cultivation of mesenchymal stem / stromal cells (MSCs) in a stirred tank bioreactor system as described herein using an impeller comprising a basket holding a packed bed composed of Fibra-Cel® Disks

[0198] Cell line and seed generation

[0199] The seed culture is prepared from 2D-expanded cells. A vial of 3 * 105-5 x 105cells is thawed and seeded in 2D and grown at 37 °C and 5 % CO2 in an incubator. Cells are passaged at 80-90 % confluency every 3 to 4 days. For bioreactor seeding, cells are detached from culture flasks, counted, pelleted and resuspended in cultivation media, and then directly seeded into the bioreactor.

[0200] Cells are cultured in pre-coated cell culture flasks using commercial MSC cultivation medium (Attachment Substrate and Mesencult ACF Plus from Stem Cell Technologies or NutriCoat Attachment Solution and MSC Nutristem XF from Sartorius) at 37 °C and 5 % CO2.

[0201] Bioreactor system preparation and setup

[0202] A DASbox bioreactor (Eppendorf) is used. Glass vessel coating (Sigmacote, Sigma-Aldrich) is performed to prevent cell attachment to said vessel. The basket of the impeller is filled with carrier material (5-5.5 g Fibra-Cel® disks) to form a packed bed, and said impeller is mounted to the stirrer shaft. The bioreactor system as is assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (see Fig. 2 for an exemplary bioreactor headplate setup). The pH and DO sensor are installed above the packed bed using a PG 13.5 port (ID 12 mm). The pH sensor is calibrated using pH 4 and pH 7 buffers before autoclaving. Feed and harvest pumps are calibrated with the feed / harvest lines and pump head tubings. PBS (particle free) is added before autoclaving for sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing are prepared as reguired and sterilized as well. The DO sensor is calibrated using 100 and 0 % oxygen, with the PBS-filled bioreactor after autoclaving.

[0203] Bioreactor system cultivation

[0204] PBS is removed, and cultivation medium (Mesencult ACF Plus from Stem Cell Technologies or MSC Nutristem XF from Sartorius) is added to the bioreactor system and equilibrated to process conditions using automated process control (pH 7.2, DO 10 %, 37 °C). Cells (e.g., MSC cell lines derived from placenta, umbilical cord, bone marrow, dental pulp, or adipose tissue) are seeded with a density of approximately 2000 cells / cm2into the bioreactor and agitation is started. The agitation rate is started with 30-50 rpm, and slowly increased to 60-80 rpm. Cultivation media is exchanged in perfusion mode, using two pumps and a level sensor to keep the volume constant. The dilution rate is controlled using off-line metabolite measurements.

[0205] Sampling and evaluation

[0206] Samples are taken from the cultivation, for metabolite analysis and microscopic imaging of the supernatant. Staining with Calcein-AM and DAPI is used to visualize live and dead cells.

[0207] Analysis of the final harvest

[0208] Disks of different parts of the packed bed are analyzed to verify homogenous conditions. Cells are stained with Calcein-AM and DAPI to check viability. CellTiter-Glo 3D assay is performed to determine the amount of viable cells per disk. Some disks are frozen for miRNA analysis.

[0209] The rest of the disks are used for dissociation of cells which is started by Versene treatment of disks after a PBS wash. Afterwards an enzyme cocktail (5 mM EDTA solution, Accutase and TrypLE Select) is applied to the disks. In order to free the cells from the fibrous mesh inside the disks, they are sonicated using an ultrasound water bath. Detached cells are counted and used for flowcytometry analysis of MSC markers.

[0210] Example 3: Cultivation of telomerized mesenchymal stem cells (MSC / TERTs) in a stirred tank bioreactor system as described herein using an impeller comprising a basket holding a packed bed composed of a three-dimensional meshed carrier material

[0211] Cell line and seed generation

[0212] The seed culture is prepared from 2D-expanded cells. A vial of 3 * 105-5 x 105cells is thawed and seeded in 2D and grown at 37 °C and 5 % CO2 in an incubator. Cells are passaged at 80-90 % confluency every 3 to 4 days. For bioreactor seeding, cells are detached from culture flasks, counted, pelleted and resuspended in cultivation media, and then directly seeded into the bioreactor.

[0213] Cells are cultured in pre-coated cell culture flasks using commercial MSC cultivation medium (Attachment Substrate and Mesencult ACF Plus from Stem Cell Technologies or NutriCoat Attachment Solution and MSC Nutristem XF from Sartorius) at 37 °C and 5 % CO2.

[0214] The applied MSC lines are stabilized by telomerization using a fragment of human telomerase (hTERT) (Wolbank et al., 2009).

[0215] Bioreactor preparation and setup

[0216] A DASbox bioreactor (Eppendorf) is used. Glass vessel coating (Sigmacote, Sigma-Aldrich) is performed to prevent cell attachment to said vessel. The basket of the impeller is filled with a three-dimensional meshed carrier material to form a packed bed, and said impeller is mounted to the stirrer shaft. The bioreactor system is assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (see Fig. 2 for exemplary bioreactor headplate setup). The pH and DO sensor are installed above the packed bed using a PG 13.5 port (ID 12 mm). The pH sensor is calibrated using pH 4 and pH 7 buffers before autoclaving. Feed and harvest pumps are calibrated with the feed / harvest lines and pump head tubings. PBS (particle free) is added before autoclaving for sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing are prepared as required and sterilized as well. The DO sensor is calibrated using 100 and 0 % oxygen, with the PBS-filled bioreactor after autoclaving.

[0217] Bioreactor system cultivation

[0218] PBS is removed, and cultivation medium (Mesencult ACF Plus from Stem Cell Technologies or MSC Nutristem XF from Sartorius) is added to the bioreactor system and equilibrated to process conditions using automated process control (pH 7.2, DO 10 %, 37 °C). Cells (e.g., MSC / TERT cell lines derived from placenta, umbilical cord, bone marrow, dental pulp or adipose tissue) are seeded with a density of approximately 2000 cells / cm2into the bioreactor and agitation is started. The agitation rate is started with 30-50 rpm, and slowly increased to 60-80 rpm. Cultivation media is exchanged in perfusion mode, using two pumps and a level sensor to keep the volume constant. The dilution rate is controlled using off-line metabolite measurements.

[0219] Sampling and evaluation

[0220] Samples are taken from the cultivation, for metabolite analysis and microscopic imaging of the supernatant. Staining with Calcein-AM and DAPI is used to visualize live and dead cells.

[0221] Analysis of the final harvest

[0222] Carrier material of different parts of the packed bed are analyzed to verify homogenous conditions. Cells are stained with Calcein-AM and DAPI to check viability. CellTiter-Glo 3D assay is performed to determine the amount of viable cells per carrier material. A part of the three-dimensional meshed carrier material is frozen for miRNA analysis.

[0223] The rest of the carrier material is used for dissociation of cells which is started by Versene treatment after a PBS wash. Afterwards an enzyme cocktail (5 mM EDTA solution, Accutase and TrypLE Select) is applied to the carrier material. In order to free the cells from the three-dimensional meshed carrier material, it is sonicated using an ultrasound water bath. Detached cells are counted and used for flowcytometry analysis of MSC markers.

[0224] Example 4: Cultivation of telomerized mesenchymal stem / stromal cells (MSCs) in stirred tank bioreactors using state of the art suspended microcarriers

[0225] The following exemplifies the use of a stirred tank bioreactor system using suspended microcarriers and an impeller for agitation, which differs from the bioreactor system as described herein comprising an agitated packed bed which holds cell carrier material.

[0226] Cell line and seed generation

[0227] The seed culture was prepared from 2D-expanded cells. A vial of 3 * 105- 5 * 105cells was thawed and seeded in 2D and grown at 37 °C and 5 % CO2 in an incubator. Cells were passaged at 80-90 % confluency every 3-4 days. For bioreactor seeding, cells were detached from culture flasks, counted, pelleted, and resuspended in cultivation media, and then directly seeded into the bioreactor.

[0228] Cells were cultured in pre-coated cell culture flasks using commercial MSC cultivation medium (Attachment Substrate and Mesencult ACF Plus from Stem Cell Technologies or NutriCoat Attachment Solution and MSC Nutristem XF from Sartorius) at 37 °C and 5 % CO2.

[0229] The applied MSC lines were stabilized by telomerization using a fragment of the human telomerase gene (hTERT) (Wolbank et al., 2009).

[0230] Microcarriers (e.g. Cytiva Cytodex 1 , Sartorius Solohill Plastic) were handled according to the manufacturer's instructions. Generally, they were suspended in solution and sterilized by autoclaving.

[0231] Bioreactor preparation and setup

[0232] A DASbox bioreactor (Eppendorf) was used. Glass vessel coating (Sigmacote, Sigma-Aldrich) was performed to prevent cell attachment. The 8-blade impeller (designed for cell culture) was mounted to the stirrer shaft. The bioreactor was assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (see Fig. 2 for exemplary bioreactor headplate setup). The pH sensor was calibrated using pH 4 and pH 7 buffers before autoclaving. Feed and harvest pumps were calibrated with the feed / harvest lines and pump head tubings. PBS (particle free) was added before autoclaving for sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing were prepared as required and sterilized as well. The DO sensor was calibrated using 100 and 0 % oxygen, with the PBS-filled bioreactor after autoclaving.

[0233] Bioreactor cultivation

[0234] PBS was removed, and cultivation medium (Mesencult ACF Plus from Stem Cell Technologies or MSC Nutristem XF from Sartorius) was added to the bioreactor and equilibrated to process conditions using automated process control (pH 7.2, DO 10 %, 37 °C). Microcarriers were transferred into the bioreactor to a density of 5 cm2 / mL and equilibrated to process conditions. Cells were seeded with a density of 2000-5000 cells / cm2into the bioreactor and agitation was started. During the attachment phase, the agitation slowly increased and afterwards continued with the just suspended agitation speed (NJS, e.g., 70 rpm at 80 mL volume). Cultivation media was aseptically added and exchanged to replace consumed cultivation media. Sampling and evaluation

[0235] Samples were taken from the cultivation, for metabolite analysis and microscopic imaging of the cells. Staining with Calcein-AM and DAPI was used to visualize live and dead cells. For microcarrier occupancy and viability data, counts of the stained cells were recorded.

[0236] Results

[0237] Fig. 10 shows microscopic images of live and dead stained (Calcein-AM / DAPI) WJ-MSC / TERT273 cells adhered to Cytiva Cytodex 1 Microcarriers (upper left and right) and Solohill Plastic Microcarriers (lower left and right), stained with Calcein-AM (live cell staining, green), after 20-24 hours of dynamic attachment and cultivation in bioreactor. Efficient cell attachment and microcarrier occupancy (fraction of carriers with at least one cell), and high viability (low DAPI staining, blue) was observed at this sampling points. The images include transmitted light (trans) microscopy to visualize the cells and microcarriers, as well as fluorescence microscopy in the green fluorescent channel. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces.

[0238] Fig. 11 shows bioreactor process data of Solohill Plastic Microcarrier-attached WJ-MSC / TERT273 in Mesencult ACF Plus Media. A media change was performed at 100 hours. Fig. 11 top panel shows the metabolite levels which were analyzed from culture suspensions using a Nova Biomedical BioProfile 100 Plus. Glucose consumption and lactate production were observed only to a small extent. Glutamate and glutamine concentrations were as expected. Fig. 11 lower panel shows the microcarrier occupancy and cell density which were analyzed and determined from microscopic images of cells stained with DAPI and Calcein.

[0239] Fig. 12 shows microscopic images of live and dead stained (Calcein-AM / DAPI) WJ-MSC / TERT273 attached to Cytiva Cytodex 1 Microcarrier in Mesencult media after 36 / 50 / 189 hours of dynamic attachment / cultivation in bioreactor (scale bar: 750 pm). To visualize cells and microcarriers, the images include fluorescence microscopy in the green fluorescent channel and overlays with transmitted light (trans) microscopy. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces. Microcarriers were added once to increase the surface area (+ 5 cm2 / mL at 50 hours) and media was exchanged (50 % at 50 hours and 180 hours) during the process.

[0240] Fig. 13 shows microscopic images of live and dead stained (Calcein-AM / DAPI) WJ-MSC / TERT273 attached to Solohill Plastic Microcarriers MSC Nutristem XF (Sartorius) media supplemented with 5 % HPL (PLT Gold, Sartorius) after 115 / 165 / 187 / 211 hours of dynamic attachment / cultivation in bioreactor (scale bar: 750 pm). To visualize cells and microcarriers, the images include fluorescence microscopy in the green fluorescent channel overlays with transmitted light (trans) microscopy and microcarriers. The Calcein and DAPI staining highlights the viability of the cells, enabling a clear distinction between the microcarriers and live cells attached to their surfaces. Microcarriers were added once to increase the surface area (+5 cm2 / mL at 50 hours) and media was exchanged (50 % at 50 hours and 180 hours) during the process.

[0241] Cells were found to attach well to microcarriers during the attachment phase of the cultivation experiment (Fig. 10).

[0242] Cells remained viable and were metabolically active for several days of the process (Fig. 11). Continuous proliferation and bead-to-bead transfer could not be achieved with the tested MSC lines (e.g., MSC / TERT cell lines derived from umbilical cord and adipose tissue) with the conditions tested. Static cell attachment, intermitted agitation and lowered / increased agitation rates, supplement addition (e.g., Gibco Revitacell, HPL, dextran sulfate, polyvinyl alcohol, anti-clumping agent) and microcarrier addition were not successful to prevent clumping of several microcarriers to each other and viability decline (Fig. 12 and Fig. 13). Cells were growing between the carriers, cells on the outside of these carrier clumps were dying, likely due to high mechanical stress. This resulted in clumps that were getting bigger with increasing process time and that could not be held in suspension anymore.

[0243] Example 5: Evaluation of the mixing performance of the bioreactor system as described herein holding a packed bed composed of Fibra-Cel® Disks

[0244] Experimental Setup

[0245] A DASbox bioreactor (Eppendorf) was used. The basket of the impeller was filled with carrier material (5-5.5 g Fibra-Cel® disks) to form a packed bed, and said impeller was mounted to a stirrer shaft. The bioreactor system was assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (Fig. 2). A pH and DO sensor were installed above the packed bed using a PG 13.5 port (ID 12 mm). The bioreactor was filled with PBS and equilibrated to process conditions (agitation, 35 °C). Without seeding cells, the mixing performance was evaluated by the addition of defined glucose shots.

[0246] Sampling and evaluation

[0247] With active agitation, 2 mL of a 25 % glucose solution was added through a short tube (on top of the liquid surface). After one minute agitation, the stirrer was turned off and samples were drawn immediately. Samples were taken from the outside using a syringe connected to a dip tube (top and bottom region), and from the inside of the packed bed using a syringe with a long needle. Glucose levels were measured using a GlucCell Meter (KDBio).

[0248] Results

[0249] A triplicate experiment at 80 rpm (standard agitation rate) and one at 30 rpm (slow mixing) indicates that the applied impeller facilitates efficient mixing. Already after one minute, the glucose shot was homogenously mixed and distributed into the packed bed (filled with Fibra-Cel® Disks).

[0250] Table 3: Glucose concentration in samples from mixing performance experiment

[0251] Example 6: Differentiation of induced pluripotent stem cells (iPSC) to neural cells in a 3D cultivation system containing Fibra-Cel® Disks

[0252] Cell line and seed generation

[0253] The seed culture was prepared from 2D-expanded cells. A vial of cells was thawed, seeded in 2D and grown at 37 °C and 5 % CO2 in an incubator. Cells were passaged at 80-90 % confluency every 3 to 4 days. For seeding on the Fibra-Cel® Disks, cells were detached from culture flasks, pelleted, resuspended in cultivation media, counted, and then directly seeded on the Fibra-Cel® Disks. Cells were cultured in 2D in pre-coated cell culture flasks using commercial iPSC cultivation medium (recombinant human Laminin-521 and Essential E8 Flex from Gibco or a self-made medium; the first 24 hours rock inhibitor was added) at 37 °C and 5 % CO2.

[0254] Preparation and setup

[0255] Either a bioreactor system, a shake flask, or any other equipment that can be used for cultivation in 3D, is prepared.

[0256] A DASbox bioreactor (Eppendorf) was prepared as follows. Glass vessel coating (Sigmacote, Sigma-Aldrich) was performed to prevent cell attachment to said vessel. The basket of the impeller was filled with carrier material (5-5.5 g Fibra-Cel® disks) to form a packed bed, and said impeller was mounted to a stirrer shaft. The bioreactor system was assembled with offgas cooler, gas filters, ports and tubes, tubings, sealing and sensors (Fig. 2). A pH and DO sensor were installed above the packed bed using a PG 13.5 port (ID 12 mm). The pH sensor was calibrated using pH 4 and pH 7 buffers before autoclaving. Feed and harvest pumps were calibrated with the feed / harvest lines and pump head tubings. PBS (particle free) was added before autoclaving for sterilization. Feed and harvest bottles, including bottles and tubing for harvest processing were prepared as required and sterilized as well. The DO sensor was calibrated using 100 and 0 % oxygen, with the PBS-filled bioreactor after autoclaving. Afterwards, PBS was removed.

[0257] A shake flask was prepared by coating (Sigmacote, Sigma-Aldrich) to prevent cell attachment and subsequent autoclaving.

[0258] 3D Cultivation

[0259] Cells were directly seeded on the Fibra-Cel® Disks with a density of 2000- 3000 cells / cm2, Essential 8 cultivation medium (Gibco) or other applicable cultivation medium containing rock inhibitor was added and the cultivation process was started. . The agitation rate / shaking speed was started with 30-50 rpm, and slowly increased to 60-80 rpm. Cultivation media is exchanged after 24 hours to neural progenitor differentiation medium (DMEM F12 Advanced medium, Neurobasal medium (mixed 1 :1), 0.5x B27 supplement minus Vitamin A, 0.5x N2 supplement, 1x Non-essential amino acids, 1x GlutaMax (all from Gibco) supplemented with 5 pM SB431542, 100 nM LDN-193189 (both Miltenyi) and 1 pM Retinoic acid (ThermoScientific). Media change or perfusion was used for up to three weeks, using manual exchange or two pumps and a level sensor to keep the volume constant. The rate of media exchange was controlled using off-line metabolite measurements.

[0260] Sampling and evaluation

[0261] Samples were taken from the cultivation, for metabolite analysis, particle measurement and microscopic imaging of the supernatant. Staining with Calcein-AM and DAPI was used to visualize live and dead cells.

[0262] Analysis of the final harvest

[0263] Disks of different parts of the packed bed were analyzed to verify homogenous conditions. Cells were stained with Calcein-AM and DAPI to check viability. Cell Titer- Glo 3D assay is performed to determine the amount of viable cells per disk. Some disks were frozen for miRNA analysis.

[0264] 10-20 disks were used for immune fluorescence (IF) of cell-specific markers (Tra-1-60, Oct 3 / 4, Nestin, MAP-2). The cells on the disks were fixed and permeabilized followed by staining with antibodies. The cell nuclei were visualized using DAPI and the stainings were analyzed under the microscope.

[0265] Results

[0266] Fig. 18 shows microscopic overlay images of cells on Fibra-Cel® Disks after 3D differentiation. Specifically, Fig. 18 shows microscopic images of cells on Fibra Cel® discs after 3D differentiation stained for specific neural (Nestin and MAP2) and iPSC markers (TRA-1-60 and Oct3 / 4). The microscopic pictures are an overlay of green (marker staining) and blue (DAPI, dead cell staining) channel images (scale bar: 750 pm).

[0267] Neural differentiation of the cells was confirmed by positive staining of the specific markers Nestin and MAP-2, and by the absence or very low signal of the iPSC markers Oct3 / 4 and TRA-1-60. The absence of Oct3 / 4 and the very low signal of TRA- 1-60 confirm the differentiation of the iPSCs. Positive staining for Nestin and MAP2 verify the differentiation to the neural pathway.

[0268] Example 7: Mixing performance of the bioreactor system as described herein holding a packed bed composed of Fibra-Cel® Disks

[0269] Fig. 19 shows the mixing performance of a bioreactor system with an impeller comprising a basket filled with Fibra-Cel® disks at 80 rpm up to 9 seconds. With active agitation, 1 mL of a 1 M NaOH solution was added through a dip tube (reaching to the bottom of the bioreactor vessel).

[0270] The verbal description of Fig. 19 is as follows. Fig. 19 shows a bioreactor system as described herein holding a packed bed composed of Fibra-Cel® Disks. The glass vessel of the bioreactor system is filled with water stained with phenol red (15 mg / L).

[0271] Considering a column represented by the liquid contained in the glass vessel of the bioreactor system, wherein the diameter (D) of said column is equal to the inner diameter of said glass vessel and the height ( / - / ) of said column is ranging from the bottom of said glass vessel to the metal ring sitting on top of the glass vessel. The height ( / - / ) of the column can be divided into 4 stacked sections. The sections are defined as follows:

[0272] • Section 1 ranges from the bottom of the glass vessel to 0.25 x H of the glass vessel;

[0273] • Section 2 ranges from 0.25 x H to 0.5 x x / - / ; • Section 3 ranges from 0.5 x H to 0.75 x / - / ;

[0274] • Section 4 ranges from 0.75 x H to H REFERENCES dos Santos, F., Campbell, A., Fernandes-Platzgummer, A., Andrade, P. Z., Gimble, J. M., Wen, Y., Boucher, S., Vemuri, M. C., da Silva, C. L., and Cabral, J. M. S. (2014). A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem / stromal cells. Biotechnology and Bioengineering, 111(6), 1116-1127.

[0275] Antebi, B., Rodriguez, L. A., Walker, K. P., Asher, A. M., Kamucheka, R. M., Alvarado, L., Mohammadipoor, A., and Cancio, L. C. (2018). Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells. Stem Cell Research and Therapy, 9(1 ), 1-15.

[0276] Wolbank, S., Stadler, G., Peterbauer, A., Gillich, A., Karbiener, M., Streubel, B., Wieser, M., Katinger, H., van Griensven, M., Redl, H., Gabriel, C., Grillari, J., and Grillari-Voglauer, R. (2009). Telomerase Immortalized Human Amnion- and Adipose-Derived Mesenchymal Stem Cells: Maintenance of Differentiation and Immunomodulatory Characteristics. Tissue Engineering Part A, 15(7), 1843- 1854.

[0277] Wang, G., Zhang, W., Jacklin, C., Freedman, D., Eppstein, L., and Kadouri, A. (1992). Modified CelliGen-packed bed bioreactors for hybridoma cell cultures. Cytotechnology, 9(1-3), 41-49.

[0278] Mizukami, A., Orellana, M. D., Caruso, S. R., de Lima Prata, K., Covas, D. T., and Swiech, K. (2013). Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system. Biotechnology Progress, 29(2), 568-572.

[0279] Pham, L. H., Vu, N. B., and Van Pham, P. (2019). The subpopulation of CD105 negative mesenchymal stem cells show strong immunomodulation capacity compared to CD105 positive mesenchymal stem cells. Biomedical Research and Therapy, 6(4), 3131-3140.

[0280] Meuwly, F., Ruffieux, P.-A., Kadouri, A., and von Stockar, U. (2007). Packed-bed bioreactors for mammalian cell culture: Bioprocess and biomedical applications. Biotechnology Advances, 25(1 ), 45-56.

Claims

CLAIMS1. A bioreactor system (1) for cultivation or expansion of adherent cells comprising one or more impellers (3) directly connected with a stirrer shaft (2), said impeller (3) comprising a basket (4) for holding a packed bed of carrier material; characterized in that said basket (4) comprises(a) two or more chambers (21), wherein the walls of the chambers (21 ) have a perforated surface (6), wherein said chambers (21) are closed by a base plate (17), and optionally a lid (20);(b) two, or more blades (5), wherein said blades (5) are attached to said basket(4); and(c) a bracket (18) at the center of at least one base plate (17) and / or of at least one lid (20), for connecting the basket (4) to the stirrer shaft (2).

2. The bioreactor system (1 ) of claim 1 , wherein the blades (5) are mounted on the outer surface of the basket (4).

3. The bioreactor system (1) of claim 1 or 2, wherein said base plate (17) and / or lid (20) have a perforated surface (6).

4. The bioreactor system (1) of any one of embodiments 1 to 3, wherein said bracket (18) comprises an elevated platform with a central hole (19), specifically said bracket (18) is a hollow cylinder.

5. The bioreactor system (1 ) of any one of claims 1 to 4, wherein the surface perforations (6) are circular, elliptical, triangular, quadrilateral, polygonal perforations, or a combination thereof, specifically of any shape geometries resulting from the union, intersection, difference, exclusion, or division of two or more shapes, or a combination thereof.

6. The bioreactor system (1) of any one of claims 1 to 5, wherein the diameter of said surface perforations (6) ranges from 0.1-10 mm, specifically said surface perforations (6) have different diameters.

7. The bioreactor system (1 ) of any one of claims 1 to 6, wherein the blades(5) are mounted on the outer surface of the base plate (17), or the lid (20).

8. The bioreactor system (1) of any one of claims 1 to 7, wherein said impellers (3) are made from a material selected from the group consisting of steel, stainless steel, aluminum, glass, ceramic, polyoxymethylene (POM), polyphenylene sulfone (PPSU), polypropylene (PP), polyether ether ketone (PEEK), ethylene- propylene-diene (EPDM), polyethylene (PE), polyamide (PA), silicone, printable resins, and bioplastics, specifically said impellers (3) are made of a corrosion resistant and sterilizable material.

9. The bioreactor system (1 ) of any one of claims 1 to 8, wherein said basket (4) has a volume of 0.01-50 L.

10. The bioreactor system of any one of claims 1 to 9, wherein the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs).

11. The bioreactor system of any one of claims 1 to 10, wherein said adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

12. The bioreactor system of any one of claims 1 to 11 , wherein said carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers, and hydrogels, and wherein said carrier material is made of a bio-compatible material that allows cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PLA), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

13. A method for cultivating, or for expanding adherent cells using a bioreactor system (1) of any one of claims 1 to 12.

14. The method of claim 13, wherein said adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically-defined media.

15. The method of claims 13 or 14, wherein the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC), immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs).

16. The method of any one of claims 13 to 15, wherein the cultivation or expansion of adherent cells is performed in batch, fed-batch, or perfusion mode.

17. The method of any one of claims 13 to 16, wherein the pH, temperature, dissolved oxygen (DO) concentration, feed or perfusion rate and agitation rate are controlled.

18. The method of any one of claims 13 to 17, wherein said carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers, and hydrogels, and wherein said carrier material is made of a bio-compatible material that allows cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PLA), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

19. A method for cultivating or for expanding adherent cells in a bioreactor system (1) of any one of claims 1 to 12, comprising the sequential steps of:(a) preparing, equipping, and assembling the bioreactor system (1) as required, specifically filling said basket (4) with a carrier material, and mounting the one or more impellers (3) to the stirrer shaft (2);(b) sterilizing said bioreactor system (1);(c) adding a cultivation medium to the bioreactor system (1);(d) seeding cells into the bioreactor system (1);(e) agitating the cultivation medium with said stirrer shaft (2) under conditions to allow cells to adhere to the carrier material;(f) exchanging the cultivation medium in perfusion mode and harvesting of conditioned medium;(g) stopping the bioreactor system (1);(h) dissociating the adherent cells from the carrier material; and(i) collecting the dissociated cells.

20. The method of claim 19, wherein the adherent cells are derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, dental pulp, or cells isolated from human urine samples, specifically the adherent cells are selected from the group consisting of mesenchymal stromal cells (MSC) and immortalized mesenchymal stromal cells, MSC / TERT, HEK293 cells, Vero cells, induced pluripotent cells (iPSCs).

21. The method of claim 19 or 20, wherein the carrier material is two- or three-dimensional, specifically wherein said carrier material is selected from the group consisting of sheet-like or meshed texture, particles, disks, fibers and hydrogels, and wherein said carrier material is made of a bio-compatible material for cell attachment of adherent cells, specifically selected from the group consisting of polyester, polypropylene, polyethylene terephthalate (PET), polyethylene (PE), printable resins, silicone, porous glass, bioplastics, polylactic acid (PLA), alginate, cellulose, collagen, fibrinogen, polyacrylamide (PA), polypeptides, and hyaluronic acid (HA).

22. The method of any one of claims 19 to 21 , wherein said adherent cells are cultured or expanded in a culture medium selected from the group consisting of serum- and human platelet lysate (hPL)-free cultivation media, preferably chemically- defined media.

23. The method of any one of claims 19 to 22, wherein the adherent cells are seeded with a density of 1000-200000 cells / cm2carrier material, specifically 1000- 100000 cells / cm2, specifically 1000-15000 cells / cm2carrier material.

24. The method of any one of claims 19 to 23, wherein the agitation is increased in a stepwise manner from 0 to 500 rpm, more specifically the agitation is increased stepwise in steps of 10-50 rpm with a final speed of 50-500 rpm.

25. Use of the bioreactor system (1) of any one of claims 1 to 12 or use of the methods of any one of claims 13 to 24 for producing viruses, extracellular vesicles(EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells.

26. A method for producing any one of viruses, extracellular vesicles (EVs), artificial cell-derived vesicles (ACDVs), or enucleated cells using the bioreactor system (1 ) of any one of embodiments 1 to 12 or using the methods of any one of embodiments 13 to 24.

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