WO2005044972A2

WO2005044972A2 - A three-dimensional carrier for culturing microbiological material

Info

Publication number: WO2005044972A2
Application number: PCT/DK2004/000771
Authority: WO
Inventors: Peter Esser
Original assignee: Nunc AS
Current assignee: Nunc AS
Priority date: 2003-11-06
Filing date: 2004-11-05
Publication date: 2005-05-19
Anticipated expiration: 2006-05-06
Also published as: WO2005044972A3

Abstract

A carrier of a biocompatible material for the culturing of anchorage dependent cells is provided. The carrier is a three-dimensional carrier for culturing microbiological material, comprising at least two substantially plane, intersecting surfaces with a substantially circular circumference. The substantially circular circumference of the intersecting surfaces imparts a substantially spherical geometry to the carrier, i.e. the carrier may be perceived to be a solid sphere or a solid polyhedron with cut-outs providing the plane surfaces. The substantially spherical geometry of the carrier allows dense packaging of carriers inside a container, such as a roller bottle, a flask, a dish, a well plate, a tray, a cell bag, an elution column, etc, whereby the growth area in the container is increased considerably. Further, the geometry of the carriers minimizes interference between surfaces of neighbouring carriers when packed and the substantially spherical geometry allows the carriers to roll.

Description

A THREE-DIMENSIONAL CARRIER FOR CULTURING MICROBIOLOGICAL MATERIAL

The present invention relates to a three-dimensional carrier for culturing microbiological material, comprising at least two substantially plane, intersecting surfaces with a substantially circular circumference.

Cell cultures are used for the production of a vast variety of products, and various methods and apparatus exist for the cultivation of cultures of bacterial and fungal cells, and cells from higher organisms.

Typically, cells are cultured in containers such as so-called roller bottles, i.e. cylindrical vessels, usually of glass, polystyrene, or polyethyleneterephthalate, which are partially filled with a liquid medium comprising the cells. The bottles are rotated slowly about their longitudinal axes, promoting aeration, while the liquid medium provides nutrients to the cells as they grow on the inner surface of the container.

For cells requiring a support for their growth (anchorage dependent cells), i.e. they must attach to a surface or a solid substratum in order to be able to grow, the relatively small surface area provided by the interior of the container limits the efficiency of conventional growth vessels.

In US 6,130,080, a roller bottle is disclosed with a rough or uneven polymeric inner cell growth surface for culturing cells. The growth surface is formed as an integral part of the roller bottle.

In WO 01/46266, chitosan beads are disclosed having relatively large and uniform pores increasing their surface area for cell growth.

It is an object of the present invention to provide a carrier for culturing biological material with an improved surface to volume ratio. It is a further object of the present invention to provide a carrier for culturing biological material that can be produced easily and at a low cost.

According to the present invention, a carrier of a biocompatible material for the culturing of anchorage dependent cells is provided. The carrier is a three-dimensional carrier for culturing microbiological material, comprising at least two substantially plane, intersecting surfaces with a substantially circular circumference.

Throughout the present specification, a three-dimensional object means an object that extends in three mutually perpendicular directions in space with dimensions in the same order of magnitude, i.e. for a three-dimensional object it is not possible to choose three mutually perpendicular directions in space where the extension in one direction is insignificant or infinitesimal in relation to the other directions, i.e. no directions can be found where the extension of the object is less than 1/10'th of the extension in the other directions.

The substantially circular circumference of the intersecting surfaces imparts a substantially spherical geometry to the carrier, i.e. the carrier may be perceived to be a solid sphere or a solid polyhedron with cut-outs providing the plane surfaces. The substantially spherical geometry of the carrier allows dense packaging of carriers inside a container, such as a roller bottle, a flask, a dish, a well plate, a tray, a cell bag, an elution column, etc, whereby the growth area in the container is increased considerably.

Further, the geometry of the carriers according to the present invention minimizes interference between surfaces of neighbouring carriers when packed. For example, when used in a roller bottle, the roller bottle is filled with carriers so that individual carriers cannot move when the bottle is rotated. Abutment of two carriers typically takes place along the thin outer circumference of the carriers leaving the plane growth surface of each carrier substantially unblocked by other carriers.

In other embodiments, it is an advantage that the substantially spherical geometry allows the carrier to roll.

Preferably, the carrier comprises at least three substantially plane, intersecting surfaces with a substantially circular circumference. In a preferred embodiment, three substantially plane and intersecting surfaces constitute the carrier. Preferably, the three axes of intersection are substantially mutually perpendicular and even more preferred, the plane surfaces are also substantially mutually perpendicular. Capillary attraction in the volume defined between neighbouring plane surfaces of a carrier makes nutrition liquid available across the entire growth surface including the inner corners of the volume.

A further embodiment of the present invention comprises five substantially plane, intersecting surfaces with a substantially circular circumference whereby the growth area may be further increased.

Further embodiments with a different number of growth surfaces may be contemplated. It is preferred that the wall thickness of the material of the carrier is minimised so that the carrier will displace as little liquid as possible while still maintaining mechanical stability of the carrier.

Preferably, the carrier is made of a material that withstands sterilization, such as, e.g., sterilization by irradiation (beta or gamma radiation), steam autoclave, ethylene oxide, chemical disinfectants, or dry heat sterilization.

In an embodiment of the invention, the carrier is made from a polymeric material that is processable by injection moulding and biocompatible, preferably hydrophilic. Examples of materials that are suitable for use in the present context are e.g., polyethylene, polypropylene, polystyrene, polycarbonate, polyurethane, polysulfone, polymethylpentene, polymethylmetacrylate, polyethyleneterephthalate, polytetrafluoroethylene, cycloolefin copolymers, or ABS (acrylonitrilbutadiene styrene). However, the examples given here only illustrate suitable materials and a person skilled in the art will know how to select other materials suitable for use as a carrier material. In a preferred embodiment, the carriers are made of polystyrene by injection moulding.

Generally, substrates need to be surface treated after moulding in order to make the surface hydrophilic and to enhance the likelihood for effective cell attachment. Surface treatment may take the form of a surface coating, but typically involves the use of directed energy at the substrate surface with the intention of generating chemical groups on the polymer surface. These chemical groups will have a general affinity for water or otherwise exhibit sufficient polarity to permit stable adsorption to another polar group. These functional groups lead to hydrophilicity and/or an increase in surface oxygen and are properties recognised to enhance cell growth. The carrier may have a diameter up to 10 mm. Preferably, the carrier walls are very thin, i.e. substantially less than the diameter of the carrier, in order for the carrier to occupy as small a volume as possible during use while still retaining the required mechanical stress to endure the impact of the surroundings, such as neighboring carriers, without breaking. In a presently preferred embodiment, the wall thickness is around 1/10'th of the carrier diameter, more preferred less than 1/10'th the carrier diameter provided that the carrier material has the required strength.

It is preferred to make the carriers small, since the smaller the carrier the easier it is to maintain the carriers in a suspension in aqueous media, i. e. with slow stirring or even without stirring. Also, the ratio between the surface area provided by the carrier and the volume occupied by the carrier increases with decreasing carrier diameter provided that the thickness of the carrier walls is kept significantly less than the carrier diameter. Thus, according to a preferred embodiment the carrier diameter is 7 mm, or less than 7 mm, such as 4 mm or 2 mm. Micro sized carriers may be provided according to the present invention with a diameter that is less than 1 mm, such as 0,75 mm, 0,50 mm, 0,25 mm, etc.

In order to provide a large carrier surface area, it is preferred that the surfaces of the carriers according to the invention are continuous surfaces with no holes or apertures making the entire area available for cell growth. When the carriers are intended for suspension in aqueous media, the carrier material of a density close to 1.0 g/ml, such as in the range from 0.90 g/ml to 1.1 g/ml, from 0.93 g/ml to 1.07 g/ml, from 0.95 g/ml to 1.05 g/ml, from 0.98 g/ml to 1.02 g/ml, is preferably selected, e.g. cycloolefin copolymers have a density of app. 1.02 g/ml and polymethylpentene has a density that is less than 1.0 g/ml. A carrier density close to or equal to the density of the nutrition liquid minimizes the need for agitation. Agitation requirements may be further decreased by utilization of small carriers. Various polymers may be mixed in order to obtain a desired density, e.g. as mentioned above.

In one embodiment the carrier material has a density that is less than and close to the density of the nutrition liquid. Thereby the carrier material tends to move towards the surface of the liquid medium, which may be desirable for growth of certain microbiological material.

In another embodiment the carrier material has a density that is larger than and close to the density of the nutrition liquid. Thereby the carrier material tends to move away from the surface of the liquid medium, which may be desirable for growth of certain microbiological material.

Preferably, cells are detached from the carrier by well-known methods, e.g. by means of trypsine or ethylenediamine tetraacetate (EDTA).

The following formulas for approximate characteristics of the carrier illustrated in Fig. 1 have been deduced, provided that wall thickness b « carrier radius r:

Surface (apart from outer edges): S = θ r² - 4br + b²)

Volume: V = Sbfar² - 4br + b²)

Packing number per litre: N_L = (100/2r)³- Displacement per litre: D = N_L ^• V Below, the features of a carrier with b = 0.06 cm and r = 0.35 cm are compared to the same features of a solid bead with the same radius for illustration of advantages of carriers according to the present invention, such as provision of a large surface area for growth of biological material with minimum displacement of liquid:

Further, the growth surface of a standard 12 cm 0 roller bottle may be increased by a factor of more than 22 by packing the bottle with carriers having these characteristics. The person skilled in the art will realise that similar advantages may be obtained in other containers for growth of biological substances, e.g. in a flask, a dish, a well plate, a tray, a tray stack, a cell bag, etc.

The term substantially circular circumference is intended to cover all shapes resembling a circle or a part of a circle, such as an elliptical shape, a parabolic shape, a hyperbolic shape, a polygonal shape, etc. However, it is important that the circumference of the substantially plane surfaces does not match or fit into the volume defined between neighbouring plane surfaces of a carrier, since a fit would make it possible for abutting carriers to engage with each other in such a way that significant parts of a surface of one carrier may be blocked by an abutting surface of another carrier. For example, if the circumference is polygonal, the angles of the polygon should be different from the angle between intersecting surfaces. Fig. 1 shows a first substantially spherical embodiment of the invention, Fig. 2 shows a second substantially spherical embodiment of the invention, Fig. 3 shows a third substantially spherical embodiment of the invention, Fig. 4 shows a fourth substantially spherical embodiment of the invention, and

Fig. 5 shows a fifth substantially polyhedral embodiment of the invention.

Fig. 1 shows a first embodiment 10 of a carrier according to the present invention. The carrier is made of polystyrene by injection moulding followed by plasma surface treatment. The carrier 10 is a three-dimensional carrier comprising three substantially plane, intersecting surfaces 12, 14, 16, each of which has a substantially circular circumference 18, 20, 22 extending 360° around the respective surface 12, 14, 16. The substantially circular circumference 18, 20, 22 of the intersecting surfaces 12, 14, 16 imparts a substantially spherical geometry to the carrier 10, i.e. the carrier may be perceived to be a solid sphere with 8 cut-outs, 4 of which are visible with reference numerals 24, 26, 32, 34, providing the plane surfaces. The three axes 28, 29, 30 of intersection are substantially perpendicular to each other. Capillary attraction in the 8 volumes, 4 of which are visible with reference numerals 24, 26, 32, 34, defined between neighbouring plane surfaces 12, 14, 16 of the carrier 10 makes nutrition liquid available across the entire growth surface including the inner corners 36 of the volume 24, 26, 32, 34.

Fig. 2 shows a second embodiment 10 of a carrier according to the present invention, corresponding to the carrier of Fig. 1 , wherein the part above the horizontal plane 14 has been turned 45° in relation to the part below the horizontal plane 14, as indicated by the arrow 11. Thus, the circumference 22 still extends 360° around the surface 14 while the other circumferences 20 extend 180° around the respective surfaces 16.

Fig. 3 shows a third embodiment 10 of a carrier according to the present invention, comprising five substantially plane, intersecting surfaces 12, 14, 16, 38, 40 with a substantially circular circumference whereby the growth area may be further increased.

Fig. 4 shows a fourth embodiment 10 of a carrier according to the present invention, comprising four substantially plane, intersecting surfaces 14, 42, 44, 46 with a substantially circular circumference. It should be noted that contrary to the embodiments shown in Figs. 1 and 2, surfaces 42, 44, 46 do not extend on both sides of their mutual intersection 48.

Fig. 5 shows a fifth embodiment 10 of a carrier according to the present invention, resembling the embodiment shown in Fig. 3 apart from the fact that the plane surfaces have polygonal circumferences. It should be noted that the carrier of Fig. 5 might be perceived to be a solid polyhedron with cut-outs providing the plane surfaces.

The carriers shown in the figures have a diameter of 7 mm and an average wall thickness of 0,6 mm. Thus, the carrier of Fig. 1 has a surface area of 1 ,8 cm² and may be packed to a density of more than 4000 carriers/litre. This means that the capacity of a common sized roller bottle having a 12 cm diameter may be increased from about 0,15 m² to more than 3,3 m², i.e. by a factor of more than 22.

In one experiment, carriers with b = 0.06 cm and r = 0.35 cm packed to a density of about 4500 carriers/litre. In this experiment, growth of a L929 cell culture was compared to growth in a stationary flask, and the effective increase in cell yield proved to correspond to an effective multiplication of the surface area of a standard 12 cm 0 roller bottle by a factor of app. 8.

Claims

I . A three-dimensional carrier for culturing microbiological material in a nutrition liquid, comprising substantially plane surfaces perceived to be formed by cut-outs in a solid, substantially spherical member having a diameter of less than 10 mm. 2. A carrier according to claim 1 , comprising substantially plane surfaces perceived to be formed by cut-outs in a solid, substantially polyhedral member.

3. A carrier according to claim 1 or 2, comprising at least two substantially plane, intersecting surfaces.

4. A carrier according to claim 1 or 2, comprising at least three substantially plane, intersecting surfaces.

5. A carrier according to claim 4, wherein one of the surfaces is substantially perpendicular to two of the at least three surfaces.

6. A carrier according to any of the preceding claims, comprising at least four substantially plane and intersecting surfaces. 7. A carrier according to any of the preceding claims, made from a material having a density approximately equal to the density of the nutrition liquid.

8. A carrier according to any of the preceding claims, made from a hydrophilic, polymeric material.

9. A carrier according to claim 8, made of a material that withstand sterilization. 10. A carrier according to claim 8 or 9, wherein the material is selected from a thermo-plastic material.

I I . A carrier according to claim 10, wherein the material is processable by injection moulding.

12. A carrier according to claim 8, wherein the material is selected from the group consisting of polyethylene, polypropylene, polystyrene, polycarbonate, polyurethane, polysulfone, polymethylpentene, polymethylmetacrylate, polyethyleneterephthalate, polytetrafluoroethylene, cycloolefin copolymers, and ABS (acrylonitrilbutadiene styrene).

13. A carrier according to claim 8, made from polystyrene. 14. A roller bottle with carriers according to any of the preceding claims. 15. A tray with carriers according to any of the preceding claims.

16. A cell bag with carriers according to any of the preceding claims.

17. A suspension with carriers according to any of the preceding claims.

18. An elution column with carriers according to any of the preceding claims.