HUE031348T2 - Eszköz sejtek aszeptikus expandálásához - Google Patents

Description

(12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12M 1104 <2006 01> C12M 1112 <2006 01> 17.08.2016 Bulletin 2016/33 C12M 1I24<2006 01> C12N 510783<2010 01> (21) Application number: 12780267.6 (86) International application number: PCT/GB2012/052587 (22) Date of filing: 19.10.2012 (87) International publication number: WO 2013/057500 (25.04.2013 Gazette 2013/17)

(54) DEVICE FOR THE ASEPTIC EXPANSION OF CELLS

VORRICHTUNG FÜR ASEPTISCHE ZELLEXPANSION DISPOSITIF POUR L EXPANSION ASEPTIQUE DE CELLULES (84) Designated Contracting States: · NEWTON, Katy Rebecca AL AT BE BG CH CY CZ DE DK EE ES Fl FR GB London W1T 6ES (GB) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO · CROMBIE, Tina, Lesley PL PT RO RS SE Sl SK SM TR London W1T 6ES (GB) • PROVAN, Andrew, Roy, MacLeod (30) Priority: 21.10.2011 US 201161550246 P Stockport SK38SR (GB) (43) Date of publication of application: (74) Representative: Hambleton, Bernadette Angelina

27.08.2014 Bulletin 2014/35 Sterling IP

Orion House (73) Proprietor: Cell Medica Limited Bessemer Road

London NW1 0QG (GB) Welwyn Garden City AL7 1HH (GB) (72) Inventors: (56) References cited: • KNAUS, Rainer, Ludwig EP-A2- 0 307 048 WO-A1-96/40876

London W1T 6ES (GB) WO-A2-2005/035728 US-A- 5 693 537 • HOLMS, Gavin US-A- 6 063 618

London W1T6ES (GB)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

Description i) a vessel comprising: [0001] The present disclosure relates to an optimised a gas permeable portion suitable for supporting system for aseptically culturing cells for therapeutic ap- cell growth and allowing delivery of gases to the plications on a commercially viable scale, methods of 5 cells during culture, and manufacturing said systems and methods of using the at least one wall adjoined to a base, wherein systems to manufacture cellular therapeutics. said vessel defines an internal volume and said [0002] WO 2005/035728 describes a system with a vessel is adapted to contain a requisite volume gas permeable portion for culturing cells. This device is of medium to support cells during culture, available from Wilson Wolf under the brand G-RexTech- 10 nology (please see the website www.wilson- ii) a vent comprising a conduit defining an interior wolf.com/page/show/67596). The main benefit of the orifice and an exterior orifice distal therefrom in fluid system is that it allows nutrients and gases to be provided communication with each other which allows balanc- to growing cells in a way so that the cells can be contin- ing of pressure during filling the vessel by allowing uously grown for periods of up to 14 days without any 15 gases to exit and during the cell-emptying process further intervention, in particular: the gas permeable allows gases to enter the interior volume, wherein membrane allows exchange of C02 and 02 and the ar- the conduit extends from the exterior of the closed rangement allows large volumes of media to be em- system through a structural feature of the system ployed which provides all the nutrients necessary for and extends into the internal volume of the vessel growth. The arrangement is shown in Figure 1. 20 and terminates therein with the interior orifice, and [0003] The main limitation of the G-Rex technology is wherein the interior orifice is arranged not to contact that it is an open manufacturing system that does not the medium in the vessel or wherein the interior or- allow the inoculation and harvesting of cells and addition ifice is covered with a liquid impermeable membrane of nutrients without exposure to the external environ- or a liquid non-return valve, such that exposure of ment. Since cellular therapeutics cannot be sterilised 25 the internal orifice to liquid is minimised and therefore post production, their manufacture has to occur under the interior orifice is not susceptible to blockage by aseptic conditions. Thus the "open" processing steps liquid during filling and emptying of liquid medium, during which the product is exposed to the external en- and wherein the exterior orifice is capable of attach- vironment have to be performed under a laminar airflow ing to a sterile filter, cabinet which is operated in a clean-room classified ac- 30 iii) a port or ports for aseptic introduction of fluid and cording to EU-GMP classification class B (US Fed. Std. cells into the vessel, 209e class 10,000, ISO 14644-1 class IS07) to prevent a port or ports for fluid to exit the system without contamination of the product with microbes and particles. exposing the system to the external environment

Facilities with such clean-room technology, for open which allows cells grown therein to exit the system processing, areexpensiveto build,operate, maintainand 35 under gravity when the system is orientated to put monitor. the cells in fluid communication with the exit port.

[0004] Equally important is the fact that the open operation steps require that only one product can be han- [0007] The system according to the present invention died at a time in the same clean-room space to minimise has significant benefits for the commercial manufacturing the risk of cross-contamination, hence, the product 40 of cells for use in therapy. In particular it is flexible and throughput is limited and this manufacturing system re- adaptable, requires low capital investment into manufac-quires multiple clean-rooms and production teams to op- turing space, and is robust and easily manufactured. It erate in parallel to achieve high volumes of production also eliminates open processing steps and therefore fur- output. For small and medium size companies as well as ther reduces the risk of contamination of the product with hospitals, the capital investment and labour costs are 45 pathogens and particles. Further, the present system is very significant on a per unit basis of production. believed to meet a currently unmet need, and represents [0005] To address this problem the present inventors a real step forward in reducing the need for expensive have provided a modified system for the culture of cells clean-room facilities for the manufacture of therapeutic that allows aseptic manufacturing of cells for use in ther- cells. apy, without the need for a clean-room environment be- so [0008] Advantageously, the present invention allows cause no open processing steps are required. the transformation of existing open system technology, such as the G-Rex system, to provide a closed system Summary of the Invention that allows the inoculation and harvesting of cells and the provision of nutrients without exposure to the external [0006] Thus the present invention provides a closed S5 environment. system suitable for aseptically culturing therapeutic cells [0009] Alternatively, a bespoke system according to comprising the present disclosure may be manufactured specifically for culturing cell therapeutics, for example a system shown in Figure 7 or 8. Detailed Description of the Invention [0010] The present disclosure also extends to methods of manufacturing the systems described herein and also [0012] A closed system as employed herein allows en- use of the systems to culture cells. try and exit of materials including liquids, cells and gases 5 without exposure to the external environment.

Brief Description of the Figures [0013] A closed system according to the present dis closure can nevertheless be employed as an open sys- [0011] tern if the end user decides not follow appropriate protocols when introducing or extracting material from the sys-

Figure 1 shows a diagrammatic representation of 10 tern. Flowever, the systems disclosed herein are ar- the commercially available G-Rex system ranged and adapted to be suitable for use as a closed for cell culture available from Wilson Wolf. system.

Figure 2 is a diagrammatic representation of a ves- [0014] Culturing cells as employed herein is intended sei comprising a lid with a vent and port co- to refer to expanding and/or differentiating cells in vitro. located therein. 15 [0015] Cell expansion as employed herein is refers to

Figure 3 is a diagrammatic representation of unitary increasing the number of the target cells in a population vessel according to the present disclosure of cells. comprising a vent and port co-located in a [0016] WO 2005/035728 incorporated herein by refer-wall of the unit. ence describes how to prepare a gas permeable vessel.

Figure 4 is a diagrammatic representation of a uni- 20 In one embodiment silicone gas permeable material is tary vessel according to the present disclo- employed. sure comprising a vent and a port located [0017] In one embodiment the base supports incorpo- opposing each other. rates the gas permeable material.

Figure 5 is a diagrammatic representation of a uni- [0018] In one embodiment the base substantially con- tary vessel of the represent disclosure 25 sists of gas permeable material i.e. the gas permeable wherein the vent and port are arranged per- material layer and the base are in fact the same entity, pendicular relative to each other. [0019] In one embodiment the gas permeable layer is

Figure 6 is a diagrammatic representation of a ves- located in a wall or other structure feature of the vessel. sei comprising a lid with a vent and portco- [0020] In one embodiment substantially all of the ves- located therein, wherein the vent extends 30 sei is prepared from a gas permeable material, for exdown into the media towards the gas per- am pie with sufficient structural strength to retain the con- meable layer. The interiororifice is covered tents during culturing. by a liquid impermeable membrane, such [0021] In one embodiment the gas permeable mem- as a gas permeable membrane to prevent brane has a surface area of, for example 5 to 200cm2, media entering the vent. 35 such as 5 to 100cm2, in particular 10, 30 or 50cm2, such

Figure 7 is a diagrammatic representation of a bell as 10cm2. shaped unitary vessel according to the [0022] A base as employed herein is a structural ele- present disclosure comprising a vent and ment of the vessel. port co-located in a wall of the unit. [0023] When cells are in the process of being cultured

Figure 8 is a diagrammatic representation of a bell 40 generally the systems will be orientated (or stood) on the shaped unitary vessel according to the base, for example the base is flat or substantially flat, present disclosure comprising a vent and Even if the system is not stood on the base, to allow air port co-located in a wall of the unit and ori- to circulate, then during culturing the base may represent entated to allow access of the media and the lowest part of the system. cells to the exit port. 45 [0024] Generally during the culturing process the cells

Figure 9 is a diagrammatic representation of how a tend to settle on and be supported by the internal surface system according to the disclosure can be of the gas permeable layer, for example in a plane which filled and harvested. is parallel to the plane of the base.

Figure 10 is a diagrammatic representation of a sys- [0025] The base in the context of the present disclo- tem according to the present disclosure so sure, for example can be understood by reference to the FigureH are detailed drawings of a system as devices shown in Figure 5 to 8, which shows the base shown in Figure 10 (1). Clearly the gas permeable layer in the base must

Figure 12 is a diagrammatic representation of how to have access to the environmental gases for the system orientate the system to harvest cells cul- to function. Thus the base may be raised above the sur- tured therein. 55 face on which the system is stood or located, to ensure access to the external gaseous environment.

[0026] Adjoined thereto as employed herein is intended to refer to the fact that one element is attached to another. [0036] In one embodimentthe lid, vent and/or ports are [0027] The vessel employed will generally be rigid or distinct materials. substantially rigid or resiliently deformable but not per- [0037] In one embodimentthe lid, vent and/or ports are manently deformable. However the vessel may comprise one material and the vessel onto which the lid is adapted portions of flexible or deformable materials. These flex- 5 to fit is the same material. ible materials may include the type of materials employed [0038] In one embodimentthe lid, vent and/or ports are in the manufacture of infusion bags. one material and the vessel onto which the lid is adapted [0028] In one embodimentthe vessel and substantially to fit is a distinct material. all the structural elements thereof are rigid. [0039] In one embodimentthe lid, vent and/or ports are [0029] The vessels and systems of the present inven- 10 distinct materials and the vessel onto which the lid is tion may be provided in a whole range of shapes and adapted to fit is a material employed in the lid orvent/port. sizes, for example derived from a cube, box, cylinder, [0040] In one embodimentthe lid, vent and/or ports are cone or pyramid. However, usually at least one area or distinct materials and the vessel onto which the lid is side of the shape will be adapted for accommodating adapted to fit is a further distinct material. vents, port or ports and/or other elements of the system. /5 [0041] In one embodimentthe a hard synthetic material

Pure shapes may be used but will not generally be em- suitable for use in aseptic manufacturing as described ployed because the shape will usually be adapted to pro- herein, such as polycarbonate, is employed to manufac-vide a bespoke vessel for the intended purpose. For ex- ture one or more of the above components, ample a cone shape may be adapted to provide a frusto- [0042] It may be advantageous from a GLP and regu- conical shape comprising a base and curved wall and a 20 latory perspective for the vessel, lid, vents and ports to top wall or lid. The top wall or lid may accommodate the be the same material. port(s) and/or vents. [0043] In one embodiment the vessel is unitary in na- [0030] In one embodiment the vessel is associated ture in that it defines a complete unitwithout a removable with or comprises a re-sealable lid, wherein the vessel or re-sealable element, such as a lid. Unitary vessels and the lid togetherform the closed system. A re-sealable 25 may, for example be moulded in one piece but unitary lid can be illustrated by reference to Figure 1 which shows as employed herein is not a reference to how the vessel a device comprising a screw lid, which is one type of re- is made but rather is a description of the function of the sealable lid. vessel and in particular that no additional structural ele- [0031] In one embodiment, the attachment of the lid to ments, such as a lid, are required to seal the vessel, the vessel is by screwing the lid through thread etched 30 [0044] In one embodiment there is provided a bell ar- in the vessel thereby allowing a seal to be created which rangement (Figure 7 and 8) wherein a structural feature protects the contents therein from contamination from opposing the base provides a concave surface in the microbes and particles in the external environment. interiorvolume. Shapes such as bells are advantageous [0032] In one embodiment a system according to the because the number of internal corners in the internal present disclosure can be provided by modifying an ex- 35 volume are minimised which may maximise the recovery isting system, for example the G-Rex unit can be modified of cells possible. The present disclosure also extends to by incorporating a vent and one or two ports, to which alternative shapes which are suitable for performing this other components of the manufacturing system can be function, in particular wherein the locations capable of aseptically connected. In one embodiment the vent and trapping cells are minimised by using "rounded surfaces", port or ports are incorporated into the lid of a G-Rex sys- 40 [0045] Thus in one embodiment the vessel is arranged tern. This provides a cost-effective way of producing a to funnel cells to the exit portwhen the system is situated closed system for culturing cell therapeutics, which can in the appropriate orientation. In one embodiment the be operated outside of a laminar air flow cabinet in a internal shape is adapted to facilitate drainage from an clean-room classified according to EU-GMP class D (US exit port when appropriately orientated.

Fed. Std. 209e class 100,000, ISO 14644-1 class ISO8) 45 [0046] The systems according to the present disclo- since the product is never exposed to the external envi- sure are arranged to allow removal of liquid and cells ronment. This greatly facilitates the aseptic manufactur- under gravity, when arranged in the required orientation, ing of the cellular therapeutic in conformance with regu- Removing the cells under gravity is advantageous be- latory requirements. cause it is simple, efficient and cost-effective. Neverthe- [0033] In one embodiment there is provided a lid ac- 50 less this process may be augmented by employing a vac- cording to the present invention for a manufacturing sys- uum, increasing the internal pressure of the system (re tem or vessel according to the disclosure, such as a G- ferred to herein as overpressure) or pumping (such as a Rex system, in particular said lid comprising a vent and/or peristaltic pump). These technologies are well known and port as discussed herein or fitting to accommodate same. may be employed in combination with the system by at- [0034] Inoneembodimentthelid.ventand/orportsare 55 tachment of the pump, vacuum or gas input to create an unitary, for example moulded. over-pressure to a relevant port or vent, as appropriate.

[0035] Inoneembodimentthelid.ventand/orportsare [0047] If desired the liquid and cells may be removed the same material, by pumping, vacuum or over-pressure without the assist- ance of gravity, even though the system is designed to of the liquid and cells to the external port using gravity or be suitable for removal of the liquid or cells gravity. pressurized systems.

[0048] A structural element as employed herein is in- [0058] The vent terminates into the internal volume is tend to refer to a base, wall, lid or other structural feature intended to refer to the fact that the vent has access to of the vessel that performs a function such as supporting, 5 the internal volume and the interior orifice may be located retaining shape and volume, holding or the like. A struc- in a structural feature of the vessel such as a wall, pro- tural element does not refer to appendages to the vessel vided that the vent is in fluid communication with the in- such as accessories, in particular ports, vents, gaskets ternal volume and that the vent is arranged such that it and the like. is generally not susceptible to blocking during harvesting [0049] Unless the context indicates otherwise interior n> of the cells, when the media and cells are less than 50% and internal are employed interchangeably herein. of the volume of the vessel.

[0050] Unless the context indicates otherwise exterior [0059] Extends into the internal volume is intended to and external are employed interchangeably herein. referto part ofthe vent conduit protruding into the internal [0051] Interior volume and interiorspace are employed space. interchangeably herein. 15 [0060] In a preferred embodiment at least part of the [0052] In one embodiment the requisite volume of me- vent physically extends and protrudes into the internal dia which the vessel is adapted to contain is an amount volume like an appendage anchored in the structural fea- that does not block the vent during culturing the cells and ture. removal of the liquid and cells. In one embodiment the [0061] In one embodiment the portion of the vent ter-volume of media is 50% or less of the internal volume, 20 minating in the internal orifice does not terminate in the such as 45%, 40%, 35%, 30%, 25%, 20% 15%, 10% or same plane as the structural element through which it is less. When the vessel is filled to this level then it may be supported, that is to say the conduit will generally pass appropriate to remove the contents under gravity. through an structural element in which it is supported and [0053] In one embodiment the maximum requisite vol- extends into the space defined by the internal volume, ume of media is 15 to 30ml, such as about 20, 21,22, 25 forexample terminating centrally in the volume as shown 23, 24, 25 or 26ml. in any one of Figures 2, 3, 5, 6, 7 and 10 to 12.

[0054] "Not susceptible to blockage by media and [0062] Located centrally as employed herein is intend- cells" as employed herein is intended to refer to the fact ed to refer to the fact that part of the vent extends from that the elements are arrangement to minimise access the structural feature ofthe vessel in which it is housed of the cells and/or media to the feature, such that it has 30 and into the space of the internal volume and is not in-a reduced propensity to becoming blocked such that is tended to be an absolute reference to the centre ofthe cannot perform its function. space per se. This feature has the benefit that during [0055] In one embodiment the volume of media is 50% filling of the vessel with media or during harvesting of or more ofthe internal volume, such as 55%, 60%, 65%, cells (in particular if the vent is co-located with the port 70%, 75%, 80%, 85%, 90% or 95%. In this embodiment 35 forentry/exit) then exposure ofthe internal orifice to liquid the maximum requisite volume of media in a system such is thereby minimised. as the G-Rex 10 is 30 to 40ml, such as about 31,32, 33, [0063] In one embodiment the vent is arranged to ter- 34, 35, 36, 37, 38 or 39ml. When the vessel is filled to minate in the volumetric centre ofthe internal volume of this level then it may be appropriate to employ a "pump" the vessel. or other system described above to assist in removing 40 [0064] Volumetric centre as employed herein isintend- the contents. The vent may be cleared of any media or ed to refer to approximately the centre of the three dicells located therein by the "pumping'Vforced extraction mensional space defined by the internal volume, process and thus the vent may become "blocked" if this [0065] In one embodiment the conduit ofthe vent exsystem is emptied under gravitation forces only. tends between 5 and 35 mm, such as about 30mm from [0056] In one embodiment the ratio of the numeric val- 45 the underside of the lid or other structural feature of the ues of the gas permeable area to the volume of the me- vessel or alternatively up from the base. dium employed in the systems is in the range 1:1 to 1:5 [0066] Inoneembodimentthe interiororifice ofthe vent respectively, for example 1:2, 1:3 or 1:4, such as 10cm2 is arranged not to contact the media contained in the gas permeable area to 20ml volume which gives a ratio vessel during use, in particular when the media and cells of 1:2. so are less than 50% of the volume of the vessel, such that [0057] The vent comprising a conduit defining an inte- the orifice and vent are not susceptible to being blocked rior orifice and exterior orifice is essentially a pipe con- by liquid or cells. necting the interior of the system to the exterior. This vent [0067] However the vent may be arranged such that it allows balancing of pressure during filling the vessel by extends into the media contained in the vessel during allowing gases to exit. During the cell emptying process 55 use. The function of the vent may be facilitated by em-the vent allows gases to enter the internal volume to fill ploying a liquid impermeable membrane, such as a gas the void that would be created by removal of the liquid permeable membrane overthe internal orifice and/or em-and cells, thereby allowing the free fluid communication ploying a liquid non-return valve in the vent.

[0068] In one embodiment the vent may comprise a moulded include, for example the same materials as the liquid non-return valve. vessel (such as thermoplastics, in particular polycar- [0069] In one embodiment the internal orifice may be bonate). In one embodiment a moulded port or ports protected by a liquid impermeable membrane, such as is/are designed such that the port or ports are suitable a gas permeable membrane. 5 for connecting to tubing, for example as shown in Figure [0070] Optionally the vent may comprise a valve to 6 where the tubing slides onto the vent or port portion control follow of gases, if desired. which is moulded.

[0071] The vent is arranged to be capable of attaching [0082] In one embodiment the port or ports are provid- to a sterile filter such as a 0.2 micron filter that prevents ed as tubing, such as flexible tubing, extending through airborne microbes and particles entering the internal vol- 10 a structural feature of the vessel and/or system. In this ume of the vessel and contaminating the system. Gen- embodiment the flexible tubing may need to be sealed erally the filtering device or element will be attached to to the structural feature by a gasket. the external orifice of the conduit forming the vent, as [0083] Tubing for use with or as a port include tubing shown in Figures 2 to 6 and 9. described supra that can be used in sterile connecting [0072] The attachment may be direct or via a coupling /5 devices for the aseptic connection of external compo- means, such as tubing. nents like e.g. sterile infusion bags.

[0073] In one embodiment the sterile filter is secured [0084] The port or ports can be located in any suitable to the vent by a fixing means such as a leur lock. location on the vessel including for example structural [0074] The vent may be prepared from any suitable features such as a wall, the base, a lid. material and may be moulded in the structural feature 20 [0085] Inoneembodimentthereisprovidedaseparate that supports it i.e. integral thereto. entrance port and a separate exit port.

[0075] In one embodiment the vent may simply com- [0086] In one embodiment there is provided one port prise flexible tubing extending through the structural fea- which functions as an entrance and exit port. ture that supports it. [0087] In one embodiment the vent is co-located with [0076] In one embodiment the vent comprises an ex- 25 a port or ports, for example in a wall or lid. terior portion extending outside the closed system which [0088] The benefit of co-locating the vent and port or is suitable for connection to flexible tubing. ports in a structural element opposite to the base, such

Suitable flexible tubing is available in many different as a lid or wall, is that no modifications to the vessel side forms, for example: wall are required. This is advantageous because vessels 30 without features in the side walls can be placed in close • a translucent tubing which contains no plasticizers, proximity to each other occupying minimal space. This latex and vinyl acetate and is also free of animal prod- also allows efficient manufacture of the vent and port or ucts, or ports within a single component of the vessel. • transfusion tubing, for example PVC transfusion [0089] The benefit of co-locating the vent and port or grade tubing such as 4mm OD PVC transfusion 35 ports in the lid means that no further modification of the grade tubing. commercially available vessel is required to convert it from an open into a closed system.

In one embodiment the tubing is silicon. [0090] In one embodiment the port employed for cells [0077] The tubing employed is such that it can be con- to exit is located proximal to a structural element, such nected aseptically to components also containing a tube 40 as wall, the edge of a lid or the like. The inventors have using a sterile tube welder, for example available from found by locating the exit port by an edge, of a wall or

Terumo Medical Corporation. structural element of the vessel then a more efficient re- 10078] The vent can be located in any suitable location covery of cells is obtained. An example the port located on the vessel including, for example in a structural feature proximal to a structural feature is shown in Figures 2 to 12. such as a wall, the base, a lid, for example in one em- 45 [0091] Having the port or ports to the side of the lid or bodiment the vent extends through the gas permeable other structural feature ensures that maximum recovery layer, which as described in detail above and may be of liquid and cells is achieved. Earlier prototypes had the located in the base of the vessel. port in the centre of the lid, and due to the surface tension [0079] In one embodiment the vent is located centrally of the liquid, it was determined that 0.5ml-0.8ml of liquid on a structural feature of the vessel, that is to say not 50 was retained in the vessel. When the port was moved to proximal to an edge of a wall, base or lid. the side, and flush with a gasket, the retention was re- [0080] In one embodiment the vent is located approx- duced to 0.1ml. imately along a central axis of the vessel, for example [0092] In addition locating the port or ports off-centre through the centre of a lid or wall and may extend to the allows the vent to be located centrally, which in at least centre of the internal volume. Centre as employed herein 55 some embodiments may minimise the exposure of the is intended to refer to approximately the midpoint of the vent to blockage by liquid. space or feature. [0093] Thus in one embodiment the port or ports, in [0081] Suitable material for the port or ports when particular the exit port is non-coaxial with a central axis of the internal volume of the vessel. [0096] Similarly empty containers such as sterile infu- [0094] In the cross-sectional figures shown herein a sion bags can be welded onto an exit port, or tubing con- double line represents a wall or barrier or similar which nected thereto, to remove the cells from the vessel asep- is closed. However a single line is not closure but is tically, also illustrated in Figure 9. present to show the shape of the feature. Thus where a 5 [0097] Both of these methods of introducing and resingle line is shown at the end of a vent or port, the same moving materials from the system aseptically are very is open. convenient, robust and practical.

Figure 2 shows a gas permeable layer (2), which forms [0098] Allowing cells to be removed (harvested) under the base (1) of vessel (4), which comprises a lid (8) co- gravity by changing the orientation of the vessel by 10 to locating a vent (6) and a port (5), wherein said vent com- to 350 degrees, for example 45 degrees or greater such as prises a conduit defining an exterior orifice capable of 90 or 180 degrees is very easy and efficient because it supporting a sterile filter said conduit extending through does not require specialised equipment and needs min- the lid and extending centrally into the interior volume imal space and expertise. defined by the vessel and terminating in the interior ori- [0099] In one embodimentthe exit port does not extend fice. In use the vessel contains media and cells which 15 above a structural feature in which it is situated into the settle on the interior surface of the gas permeable layer. internal volume more than 0.15mm. The later ensures

Figure 3 shows a similar arrangement to that of Figure maximum recovery of cells. 2 but is characterised by the vessel being a single unit [0100] The port or ports may be fitted with a valve without a re-sealable lid. The port (5) and vent (6) are and/or membrane or the like to control flow of materials co-located in a wall (a structural element) of the vessel. 20 therethrough.

The conduit of the vent extends as a protrusion into the [0101] Suitable gaskets and seals may be required deinternal volume of the vessel. pending on the exact construction of the system.

Figure 4 shows a similar arrangement to Figure 3 but [0102] A suitable material for manufacturing the vessel wherein the port (5) and vent (6) are arranged to oppose and structural elements thereof will generally be required each other. 25 to be compliant with one or more of the standards: EL)

Figure 5 shows a similar arrangement to Figures 3 and Food Approval : EL) Directive 2002/72/EC; USP <88>, 4 but wherein the port (5) and the vent (6) are arranged biological reactivity test in vivo, class VI; USP <87>, biperpendicular to each other. ological reactivity test in vitro; USP <661 >, physicochem-

Figure 6 shows a gas permeable layer (2), which forms ical test - plastics; EP monograph 3.2.2, plastic contain- the base (1) of vessel (4), which comprises a lid (8) co- 30 ers &amp; closures for pharmaceutical use; Biological tests locating a vent (6) and a port (5), wherein said vent com- according to ISO 10993-external communicating devic- prises a conduit defining an exterior orifice capable of es; for indirect blood contact for a prolonged period, supporting a sterile filter said conduit extending through [0103] In one embodimentthe vessel and/or structural the lid and extending centrally into the interior volume, elements of the systems are manufactured from a suit- defined by the vessel, towards the base and into the me- 35 able thermoplastic such as polycarbonate, dia (3). The interior orifice is protected by a gas perme- [0104] The ports and vents may be incorporated into able membrane (9) which prevents blockage of the vent the system by thermal soldering, especially when a by liquid. closed system is being created from a commercially

Figure 7 shows a unitary bell shaped vessel (4) arranged available product, such as G-Rex. with the port (5) (which has a dual function of the entrance 40 [0105] I n one embodiment a modified lid will be mould- and exit port) and the vent (6) co-located in a structural ed in one piece with appropriate connections for tubes feature of the vessel opposing the base comprising a gas and/or filter, which will be attached afterward, permeable layer. In this Figure the vent (6) and port (5) [0106] In one embodiment the features and structural are shown with the exterior portions connected to flexible functionality, such as ports and vents, will be created tubing. 45 when moulding the vessel.

Figure 8 shows a system of Figure 7 orientated to put [0107] Structural functionality as employed herein is the liquid media and cells in communication with the exit intended to refer to features which perform a function, port (5), wherein the cells may exit the port under gravity. for example as a vent or port and/or which is suitable for

The dimensions of the Figure herein are by way of ex- attaching accessories to, such as tubing or the like, ample only and are not intended to be limiting. so [0108] The systems according to the disclosure may [0095] Sterile containers, such as infusion bags, con- be gamma irradiated for sterilisation. taining materials such as a media and/or cells can be [0109] Generally the system will be delivered to end welded to an entrance port or tubing connected thereto users in a sealed bag in a sterile form, for example along using known technology to introduce media aseptically with a certificate of sterility. A system in this form will into the system. This is illustrated diagrammatically in 55 generally have a shelf-life of about 1 year or more.

Figure 9. Sterile welding techniques and technology are [0110] There is also provided a process of manufac- well known in the industry and will not be discussed fur- turing a system according to the present disclosure com- ther here. prising the step of moulding the vessel, and optionally fitting the ports and vents thereto. ually.

[0111] In one embodiment the holes to accommodate [0123] In one embodiment the cells are washed em- a vent and/or ports are created by thermal probes rather ploying an automated system, for example a Sepax®sys- than by drilling, thereby minimising the amount of con- tern available from Biosafe. tamination generated and ensuring the final product is 5 [0124] In one embodiment, for example after washing, suitable for use to prepare a therapeutic product. the cells are counted.

[0112] In one embodiment further components such [0125] In one embodiment, for example after counting, as gaskets and/or valves and/or filters are fitted in the a therapeutic amount of cells (i.e. one or more doses of manufacturing process. cells) is selected.

[0113] In one embodiment the manufacturing process 10 [0126] In one aspect the harvested cells are enclosed comprises the further step of sterilising the system, for into a suitable container, for example an infusion bag, example using gamma irradiation and aseptically sealing optionally along with one or more pharmaceutically ac- the unit in one or multiple bags or containers. ceptable preservatives or excipients, for storage.

[0114] In one embodiment one or more steps of the [0127] Suitable excipients include DMSO, for example manufacturing process are performed in a clean-room 15 10% DMSO. complying with standards to EU-GMP class D (US Fed. [0128] In one embodiment the container is transported

Std. 209e class 100,000, ISO 14644-1 class IS08). In to the location of a patient. one embodiment each system together with filters, gas- [0129] In embodiment the content of the container is kets, tubes and connections and before sterilisation the administered to a patient parenterally, particularly intra- assembled system will be subject to a pressure hold test 20 venously. with compressed air according to methods known to [0130] In the context of this specification "comprising" those skilled in the art, for example pressure testing of is to be interpreted as "including". 0.5 bar ±0.05 bar for greater than 2mins. During this [0131] Aspects of the invention comprising certain el- time, the pressure will not drop from its recorded start ements are also intended to extend to alternative em- value by less than 0.05 bar. 25 bodiments "consisting" or "consisting essentially" of the [0115] In one embodiment there is provided a method relevant elements, of modifying an existing cell-culture system to provide a closed system according to the invention. The modification may employ one or more of the manufacturing steps Claims defined above. 30 [0116] In one embodiment a vessel according to the 1. A closed system suitable for the aseptic culture of disclosure, such as a modified G-Rex vessel and/or a therapeutic cells comprising: vessel with a gas permeable membrane of about 10cm2 will typically be filled to 10 to 25ml final volume with cells (i) a vessel comprising: and culture medium, and will be incubated at 37°C until 35 the cells are ready for harvesting. Having this the vessel a gas permeable portion suitable for sup- may be filled with up to 40mls as discussed above. porting cell growth and allowing delivery of [0117] Thus a method of introducing cells aseptically gases to the cells during culturing, and to a closed system according to the disclosure followed at least one wall adjoined to a base, by incubating the cells at an appropriate temperature and 40 wherein said vessel defines an internal vol- for an appropriate period is provided. umeforcontainingarequisitevolumeofme- [0118] In a further aspect, a method of aseptically har- dium to support a cell culture, vesting therapeutic cells from a closed system according to the disclosure by aseptically joining a receptacle to an (ii) a vent comprising a conduit defining an inte- exit port of the system and harvesting the cells into re- 45 rior orifice and an exterior orifice distal therefrom ceptacle under gravity is provided. in fluid communication with each other which al- [0119] In a further aspect, a method of aseptically har- lows balancing of pressure during filling the ves- vesting therapeutic cells from a closed system according sei by allowing gases to exit and during the cell- to the disclosure by aseptically joining a receptacle to an emptying process allows gases to enter the inexit port of the system and harvesting the cells into re- 50 terior volume, ceptacle employing a pump is provided. wherein the conduit extends from the exterior of [0120] In one embodiment the receptacle into which the closed system through a structural feature the harvested cells are transferred is a 600ml bag, some- of the vessel and extends into the internal vol- times referred to in the field as a "transfer pack". ume of the vessel and terminates therein with [0121] In one embodiment after harvesting the cells 55 the interiororifice, and wherein the interior orifice are washed, for example with a solution comprising hu- is arranged not to contact the medium in the ves- man serum albumin, saline or similar. sei orwherein the interiororifice is covered with [0122] In one embodiment the cells are washed man- a liquid impermeable membrane or a liquid non- return valve, such that exposure of the internal of nutrients for culturing the cells, wherein step orifice to liquid is minimised and therefore the a may be performed before, afteror concomitant interior orifice is not susceptible to blockage by with step b, liquid during filling and emptying of liquid medi- c. culturing the cells to expand the target T cell urn, and 5 population, and wherein the exterior orifice is capable of attach- d. aseptically harvesting the cells into a suitable ing to a sterile filter, container. (iii) a port or ports for aseptic introduction of fluids and cells into the vessel, 11. A method according to claim 10, which comprises a port or ports forfluids to exit the system without 10 the further step of washing the expanded cell popu-exposing the system to the external environ- lation after harvesting, mentwhich allows cells grown therein to exit the system under gravity when the system is őrien- 12. A method according to claim 10 or 11, which com-tated to put the cells in fluid communication with prises a further step of counting the expanded cell the exit port. 15 population after harvesting. 2. A system according to claim 1, wherein the vessel 13. A method according to any one of claims 10 to 12, is unitary. which comprises a further step of separating a ther apeutic dose of the cells into a suitable container. 3. Asystem according to claim 2, wherein the structural 20 feature of the vessel through which the conduit ex- 14. A method according to any one of claims 10 to 13, tends is a wall of the vessel. wherein an excipient such as DMSO is added to the harvested cells. 4. A system according to claim 2 or 3, wherein part of the vessel is arranged to funnel cells to the exit port 25 15. Use of the system of any one of Claims 1 to 9 to when the vessel is in an appropriate orientation. aseptically culture therapeutic cells. 5. Asystem according to any one of claims 1 to 4 wherein said vent and said ports are located in the same Patentansprüche structural feature of the vessel, for example located 30 opposing the base. 1. Geschlossenes System, das sich fürdas aseptische

Kultivieren von therapeutischen Zellen eignet, das 6. A system according to any one of claims 1 to 5, umfasst: wherein the exit port is non-coaxial with a central axis of the internal volume of the vessel. 35 (i) ein Gefäß, das umfasst: 7. A system according to any one of claims 1 to 6, einen gasdurchlässigen Abschnitt, derda- wherein the exit port is located in a structural feature zu geeignet ist, ein Zellwachstum zu unter- and the port extends 0.15mm őrless into the internal stützen und eine Abgabe von Gasen an die volume. 40 Zellen während des Kultivierens zu ermög lichen und 8. A system according to any one of claims 1 to 7, zumindesteineWand.dieaneineBasisan- wherein the exterior orifice is attached to a sterile grenzt, filter. wobei das Gefäß ein inneres Volumen zur 45 Aufnahme eines erforderlichen Volumens 9. A system according to any one of claims 1 to 8, eines Mediums definiert, um eine Zellkultur wherein the conduit of the vent extends between 5 zu unterstützen, and 35 mm into the internal volume of the vessel, such as 30 mm from the structural feature of the ves- (ii) einen Luftdurchlass, der eine Leitung umsei. so fasst, eine innere Öffnung und eine distal davon angeordnete äußere Öffnung definierend, die 10. A method for aseptic culture oftherapeutic cells com- miteinander in Fluidverbindung stehen, der das prising the steps of Ausgleichen von Druck während des Befüllens des Gefäßes ermöglicht, indem er das Austre- a. aseptically adding to a closed system defined 55 ten von Gasen ermöglicht und während des Zel- in any one of Claims 1 to 9 a cell population for lentleerungsprozesses das Eintreten von Ga- culturing sen in das innere Volumen ermöglicht, b. aseptically adding to said system a nutrient wobei sich die Leitung vom Äußeren des ge- schlossenen Systems durch ein strukturelles streckt, wie z. B. 30 mm vom strukturellen Merkmal

Merkmal des Gefäßes erstreckt und sich in das des Gefäßes, innere Volumen des Gefäßes erstreckt und darin mitderinneren Öffnung endet, und wobeidie 10. Verfahren zum aseptischen Kultivieren von thera-innere Öffnung so ausgelegt ist, dass sie das 5 peutischen Zellen, das die Schritte umfasst: Medium im Gefäß nicht berührt, oder wobei die innere Öffnung mit einer flüssigkeitsdichten a. aseptisches Hinzufügen einer Zellpopulation

Membran odereinem Flüssigkeits-Rückschlag- zum Kultivieren in ein geschlossenes System, ventil bedeckt ist, so dass die Flüssigkeitsexpo- wie in einem der Ansprüche 1 bis 9 definiert, sition der inneren Öffnung minimiert wird und 10 b. aseptisches Hinzufügen eines Nährstoffs von som it die innere Öffnung nicht für eine Blockade Nährstoffen zum Kultivieren der Zellen zum Sys- durch Flüssigkeit während des Befüllens und tem, wobei Schritt a vor, nach oder begleitend

Leerens von flüssigem Medium anfällig ist, und mit Schritt b durchgeführt werden kann, wobei die äußere Öffnung in der Lage ist, mit c. Kultivieren der Zellen, um die Ziel-T-Zell-Po- einem sterilen Filter verbunden zu werden, 15 pulation zu expandieren, und (iii) ein oder mehrere Löcher zum aseptischen d. aseptisches Ernten der Zellen in einen geeig-

Einbringen von Fluiden und Zellen in das Gefäß, neten Behälter, ein oder mehrere Löcher, über die Fluide aus dem System austreten können, ohne das Sys- 11. Verfahren nach Anspruch 10, das den weiteren tem der Außenumgebung auszusetzen, wo- 20 Schritt des Waschens der expandierten Zellpopula-durch möglich wird, dass darin gezüchtete Zel- tion nach dem Ernten umfasst, len durch Schwerkraft aus dem System austreten, wenn das System so ausgerichtet ist, dass 12. Verfahren nach Anspruch 10 oder 11, das einen wei- die Zellen mit dem Austrittsloch in Fluidverbin- teren Schritt des Zählens der expandierten Zellpo- dung gelangen. 25 pulation nach dem Ernten umfasst. 2. System nach Anspruch 1, wobei das Gefäß unitär ist. 13. Verfahren nach einem der Ansprüche 10 bis 12, das einen weiteren Schritt des Abtrennens einer thera- 3. System nach Anspruch 2, wobei das strukturelle peutischen Dosis der Zellen in einen geeigneten Be-

Merkmal des Gefäßes, durch das sich die Leitung 30 hälter umfasst. erstreckt, eine Wand des Gefäßes ist. 14. Verfahren nach einem der Ansprüche 10 bis 13, wo- 4. System nach Anspruch 2 oder 3, wobei ein Teil des bei ein Exzipient wie z. B. DMSO zu den geernteten

Gefäßes so ausgelegt ist, dass Zellen trichterförmig Zellen hinzugefügt wird. zum Austrittsloch geleitet werden, wenn sich das Ge- 35 fäß in einer entsprechenden Ausrichtung befindet. 15. Verwendung des Systems nach einem der Ansprü che 1 bis 9 zum aseptischen Kultivieren von thera- 5. System nach einem der Ansprüche 1 bis 4, wobei peutischen Zellen, der Luftdurchlass und die Löcher im gleichen strukturellen Merkmal des Gefäßes angeordnet sind, z. 40 B. der Basis gegenüberliegend. Revendications 6. System nach einem der Ansprüche 1 bis 5, wobei 1. Systéme fermé adapté á la culture aseptique de cel- das Austrittsloch nicht koaxial mit der Mittelachse lules thérapeutiques, ledit systéme comprenant: des inneren Volumens des Gefäßes vorliegt. 45 (i) un recipient comportant: 7. System nach einem der Ansprüche 1 bis 6, wobei das Austrittsloch in einem strukturellen Merkmal an- une partié perméable aux gaz qui est adap- geordnetistundsichdasLochO,15mmoderweniger tée pourfavoriser la croissance cellulaire et in das innere Volumen erstreckt. 50 permettre la délivrance des gaz aux cellules pendant la culture, et 8. System nach einem der Ansprüche 1 bis 7, wobei au moins une paroi attenante ä une base, die äußere Öffnung mit einem sterilen Filter verbun- ledit recipient définissant un volume inté- den ist. rieurdestiné á contenir un volume de milieu 55 nécessaire pour favoriser une culture de 9. System nach einem der Ansprüche 1 bis 8, wobei cellules, sich die Leitung des Luftdurchlasses zwischen 5 und 35 mm in das innere Volumen des Gefäßes er- (ii) un évent comportant un conduit définissant un trou intérieur et un trou extérieur, distal du 8. Systémeselon l’unequelconquedesrevendications trou intérieur, qui sont en communication fluidi- 1 ä 7, dans lequel le trou extérieur est fixé a un filtre que l’un avec l’autre, ce qui permet d’équilibrer stérile. la pression pendant le remplissage du récipient tout en permettant aux gaz de sortir et ce qui 5 g. Systémeselon l’unequelconquedesrevendications permetauxgazd’entrerdans le volume intérieur 1 ä 8, dans lequel le conduit de l’évent s’étend sur pendant le processus de vidage des cellules, unelongueurcompriseentre5et35mmjusquedans le conduit s’étendant de l’extérieur du Systeme le volume intérieur du récipient, par exemple sur 30 fermé a travers un élément structurel du réci- mm ä partirde l’élément structurel du récipient. pient et s’étendant jusque dans le volume inté- 10 rieur du récipient pour déboucher ä l’intérieur 10. Procédé de culture aseptique de cellules thérapeu-par le trou intérieur, et le trou intérieur étant dis- tiques comprenant les étapes consistant ä posé de fagon á éviter le contact avec le milieu dans le récipient ou le trou intérieur étant recou- a. ajouter de maniére aseptique ä un systéme vert d’une membrane imperméable aux liquides 15 fermé défini selon l’une quelconque des reven- ou d’un clapet de retenue de liquide de fagon á dications 1 a 9 une population de cellules en vue minimiserrexpositiondutrou intérieurau liquide de la culture et par conséquent de telle sorté que le trou in- b. ajouter de maniére aseptique audit systéme térieur n’est pas susceptible d’etre bloqué par un nutriment parmi des nutriments utilisés pour le liquide pendant le remplissage et la vidage du 20 |a culture des cellules, l’étape a peut étre effec- milieu liquide, et tuée avant ou aprés l’étape b ou de fagon con- le trou extérieur pouvant étre fixé á un filtre sté- comitante, rile, c. cultiver les cellules en vue de l’expansion de (iii) un ou plusieurs orifices d’introduction asep- la population de cellules T cibles, et tique de liquides et de cellules dans le récipient, 25 d. récolter de maniére aseptique des cellules un ou plusieurs orifices de sortie de fluides du dans un récipient approprié. systéme sans que le systéme sóit exposé á l’en- vironnement extérieur, ce qui permet aux cellu- 11. Procédé selon la revendication 10, qui comprend les cultivées á l’intérieur de sortir du systéme l’étape supplémentaire de lavage de la population par gravité lorsque le systéme est orienté de fa- 30 de cellules expansée aprés la récolte. gon é mettre les cellules en communication flui- dique avec l’orifice de sortie. 12. Procédé selon la revendication 10 ou 11, qui com prend en outre une étape de comptage de la popu- 2. Systéme selon la revendication 1, dans lequel le ré- lation cellulaire expansée aprés la récolte. cipient est unitaire. 35 13. Procédé selon l’une quelconque des revendications 3. Systéme selon la revendication 2, dans lequel l’élé- 10 a 12, qui comprend en outre une étape de sépa- ment structurel du récipient, a travers lequel le con- ration d’une dose thérapeutique des cellules dans duit s’étend, est une paroi du récipient. un récipient approprié. 40 4. Systéme selon la revendication 2 ou 3, dans lequel 14. Procédé selon l’une quelconque des revendications

une partié du récipient est adaptée pour canaliser 10 á 13, dans lequel un excipient tel que du DMSO les cellules ä l’orifice de sortie lorsque le récipient est ajouté aux cellules récoltées. est dans une orientation appropriée. 45 15. Utilisation du systéme selon l’une quelconque des 5. Systémeselon l’une quelconque des revendications revendications 1 á 9 pour cultiver de maniére asep- 1 a 4, dans lequel ledit évent et lesdits orifices sont tique des cellules thérapeutiques. situés dans le mérne élément structurel du récipient, par exemple en face de la base. 50 6. Systéme selon l’une quelconque des revendications 1 á 5, dans lequel l’orifice de sortie n’est pas coaxial á un axe central du volume intérieur du récipient. 7. Systémeselon l’une quelconque des revendications 55 1 á 6, dans lequel l’orifice de sortie est situé dans un élément structurel et l’orifice s’étend sur 0,15 mm ou moins jusque dans le volume intérieur.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • WO 2005035728 A [0002] [0016]

Claims (7)

1. ESZKÖZ SEJTEK ASZEPTIKUS EXPANPALÁSÁHOZ SZABADALMI IGÉNYPONTOK

   1, Gyógyászati sejtek aszeptikus tenyésztésére alkalmas zárt rendszer, amely magában foglal: (!) egy edényt, amely magában foglal: egy, a sejtnövekedés fenntartására alkalmas és gázoknak a tenyésztés során a sejtekhez történd szállítását lehetővé tévő gázátereszté részt, és legalább egy tatet, amely egy stephoz van hozzákapcsolva, amely edény meghatároz egy belső volument egy sejtkultúra fenntartásához szükséges mennyiségű közeg befogadására* (is) egy szellőzőt, amelyet egy vezeték képez, amely meghatároz egy belső nyílást és egy ahhoz képest disztálís, külső nyílást, amelyek folyadék-összeköttetésben vannak egymással, an# a gázok kilépésének megengedése révén tehetővé teszi a nyomás kiegyenlítését az edény töltése alatt, és a sejt-ürítési folyamat alatt megengedi a gázok belépését a belső volumenbe, amely vezeték a zárt rendszeren kívülről, az edény egy szerkezeti elemén keresztül terjed ki, benyúiik az edény belső volumenébe, és ott a belső nyílásban végződik, továbbá a belső nyílás ágy van elhelyezve, hogy ne érintkezzen az edényben iévö közeggel, vagy a belső nyílást egy folyadékot át nem eresztő membrán vagy egy folyadék visszacsapó szetep fedi, úgyhogy a belső nyílás minimális mértékben van kitéve folyadéknak, ezért a belső nyílást nem tudja a folyadék eizárni a folyékony közeg betöltése és kiürítése alatt, ezenkívül a külső nyílást csatlakoztatni lehet egy steril szűrőhöz, (Ili) egy portot vagy portokat folyadékoknak és sejteknek az edénybe történő aszeptikus bejuttatására, egy portot vagy portokat folyadékok kiléptetésére anélkül, hogy a rendszer ki tenne téve a külső környezetnek, ami lehetővé teszi, hogy az ott növekvő sejtek a gravitáció hatására lépjenek ki, amikor a rendszer olyan helyzetbe van állítva, hogy a sejtek folyadék-összeköttetésbe kerüljenek a kilépő porttal,
2. 2, Az 1. igénypont szerinti rendszer, amelyben az edény egységes, 3, A 2, igénypont szerinti rendszer, amelyben az edénynek azt a szerkezeti etemét, amelyen keresztül a vezeték kiterjed, az edénynek egy fala képezi, 4, A 2, vagy a 3. igénypont szerinti rendszer, amelyben az edénynek egy része úgy van elhelyezve, hogy tőiesérszerüen a kilépő porthoz vezesse a sejteket, amikor az edény megfelelő helyzetbe van állítva,
3. 5. Az 1-4, igénypontok bármelyike szerinti rendszer, amelyben a szellőző és a portok az edénynek ugyanabban a szerkezeti elemében, például az alappal szemközt vannak elhelyezve. 6 Az i"S, Igénypontok bármelyike szerinti rendszer, amelyben a kilépő port nem koaxiális az edény belső volumenének központi tengelyével. ?, Az l-δ, igénypontok bármelyike szerinti rendszer, amelyben a kilépő port egy szerkezeti elemben van elhelyezve, és a port 0,15 mm-re vagy annál kisebb távolságban nyúlik be a belső volumenbe,
4. 8, Az 1-7. Igénypontok, bármelyike szerinti rendszer, amelyben a külső nyílás csatlakoztatva van egy steril szűrőhöz,
5. 9, Az 1~$. igénypontok bármelyike szerint* rendszer, amelyben a szellőző vezetéke 5 és 35 mm közötti távolságban nyúlik be az edény belső volumenébe, az edény szerkezeti elemétől például 30 mm*re,
6. 10, Eljárás gyógyászati sejtek aszeptikus tenyésztésére, amely eljárás a következő lépéseket foglalja magábam a) aszeptikusán hozzáadunk egy, az l~9. igénypontok bármelyike szerinti zárt rendszerhez egy sejtpopulációt tenyésztésre, b) aszeptikusán hozzáadunk a rendszerhez tápanyagot vagy tápanyagokat a sejtek tenyésztéséhez, és az a) lépés végezhető a b) lépés előtt, után vagy azzal együtt, e> tenyésztjük a sejteket a T céi-sejtpopuláció expandálása céljábéí, és d) aszeptikusán begyőjtjűk a sejteket egy megfelelő konténerbe. ÍL A 10. igénypont szerinti eljárás, amely magában foglalja azt a további lépést, hogy a begyűjtés után az expandálódéit sejtpopulációt mossuk. 12. A 10. vagy a 11. igénypont szerinti eljárás, amely magában foglalja azt a további lépést, hogy a begyűjtés után az expandáíódott sejtpopulációt megszámláljuk. 13. A 10-12;, igénypontok bármelyike szerinti eljárás, amely magában foglalja azt a további lépést, hogy a sejtek gyógyászati adagját egy megfelelő konténerbe, különítjük, 14. A 10-13, igénypontok bármelyike szerinti eljárás, amelyben a begyűjtött sejtekhez hozzáadunk egy kötőanyagot, például dimetü-szulfoxidot
7. 15. Az l-S, Igénypontok bármelyike szerinti rendszer alkalmazása gyógyászati sejtek aszeptikus tenyésztésére,