DE3341771A1 - Microcarriers for use as growth sites for anchorage-dependent cells, and process for their preparation - Google Patents

Abstract

A microcarrier for anchorage-dependent cell culture comprises and consists preferably essentially of a spherical substrate of polymeric material with a density of approximately 1 g per cubic centimetre so that it essentially floats in an aqueous culture medium, and of a thin (less than 1 mu m) coating layer of silicate glass. The silicate glass coating layer is preferably applied to a polymer "precursor" in a radiofrequency atomisation process. An intermediate coating layer of magnetic material can be applied before the silicate glass layer. In this way it is possible for the microcarriers to be easily removed from culture media by the action of a magnetic field.

Description

DESCRIPTION:

The invention relates to a microcarrier for use as a growth site for anchorage-dependent cells in an aqueous cell culture and a method for its production.

In the cultivation of anchorage-dependent cell tissue cultures it is known, the conventional drum bottles and Petroschalen by so-called Replace microcarriers, which have an enlarged area for cell attachment Provide. According to US Pat. No. 4,189,534, for example, microcarriers used in the form of solid plastic beads. However, it has been found that such plastic microcarriers to promote cell adhesion alternating electrically require charged surface constituents. This variety can be found in the It is difficult to control production quantitatively.

If not properly controlled, it is for some species toxic from cell cultures. It is also difficult to remove some types of cells from the surface remove the plastic balls.

It is also known to use solid glass beads as cell microcarriers use. In addition to the problems already mentioned above, there is an essential one Disadvantage of known microcarriers, in particular of solid plastic beads or glass, in that the density of the microcarriers is very difficult to control and must be matched to that of the respective culture medium. Conventional cell culture media are aqueous and have densities generally in the range between 1.03 and 1.09 g per cubic centimeter. Silicate glass beads, which have favorable surface properties typically have a density of the order of 2.3 grams per cubic centimeter depending on the glass composition. When the weaning and the compaction the microcarrier in the growth medium should be avoided, which would hinder cell growth the culture medium must be stirred or otherwise kept in constant motion will. However, vigorous movement is destructive even to many types of cells. A general overview of the use of microcarriers in cell culture organisms can be found in "Third General Meeting of ESACT", Oxford, 1979, Develop. Biol. Standard, 46, p.p.

109-294 (S. Karger, Basel, 1980).

In U.S. Patent Application No. 332,377, those described above are disclosed and other difficulties are addressed by using hollow glass "precursor" microspheres can be formed from silicate glass. Thereafter, the density of these "precursor" microspheres is determined tailor-made in a post-forming etching process so that they exactly match the density of the corresponds to the desired aqueous growth medium.

This procedure has proven to be very successful in overcoming it of difficulties with surface charge and buoyancy. The number however, separate operations makes a cost reduction desirable.

The object of the present invention is to create a microcarrier, whose density comes very close to that of typical cell culture media and the has the desired surface properties of silicate glass, but cheaper is to be produced as a known microcarrier with similarly good properties.

This task is described in the characterizing part of the main claim Invention solved; advantageous developments of the micro-carrier are in the claims 2 and 3 indicated.

The microcarrier according to the invention can be very easily removed from the culture media remove and is particularly good for rapid removal using automated Procedure suitable.

The object of the present invention is also to provide a method for Specify the production of such microcarriers.

This task is described by the characterizing claim 4 Invention solved; advantageous developments of the manufacturing method according to the invention are given in claims 5 to 7.

According to the invention, the microcarriers are produced in that initially a spherical "precursor" is fabricated from polymer material, the density of which is im essentially the same as that of the desired aqueous growth medium is (approximately 1 g per cubic centimeter). Thereafter, the polymer "precursor" will use covered with a thin layer of silicate glass. The thin layer of glass changed the effective density of the "precursor is negligible, but gives it the considerable advantages of the glass adhesive surface, which is toxicity and lightness reached, with which cultures can be removed without significant damage.

When the microcarriers are provided with a composition that at least partially made of magnetic material, they can easily be through a magnetic field attracted and removed from the culture medium.

A preferred embodiment is described in more detail below.

First, according to a known method, a spherical "precursor" made of polymer material. For example, a film-forming polymer in a suitable solvent and then dissolved in the top portion of a heated chamber or an oven in the form of small droplets are sprayed. if the droplets fall under the influence of gravity within the heated chamber, the solvent evaporates quickly; a polymer skin or shell is formed. Such a method and the associated apparatus are described in US patent specification 2,797,201. The nominal precursor size and density are called Function of the polymer / solvent concentration, the drying temperature and the Drop size regulated. The drop size is typically in the range between 5 and 500 / µm. The drying temperatures are in the range between 200C and 5000C. The completed "precursor" can be taken up dry on the underside of the chamber, cleaned and to the desired size range, for example between 106 and 200 / u, to be sieved.

The polymer "precursor" can also be made as a solid bead by spraying molten polymer material and cooling it. Included will place a drop generator or other known device in a free fall zone directed in such a way that the droplets are cooled and solidified during free fall and then be collected. Alternatively, solid pieces (frit) made of polymer material can be used of a certain size are fed into a tower furnace, where they are melted and transformed into spheres during free fall.

des The polymer material / "precursor" is chosen so that the desired density in the desired size range and coatability with Glass shows that, for example, polystyrene and polyethylene are suitable materials. The "precursor" can be solid, hollow or porous. Depending on Precursor material and the method used in coating, a surface treatment of the "Forerunner" can be recommended. In this way it is easier to get along with cover the silicate glass. Precursor diameter in the range between 50 µm and 500 µm and densities in the range between 1.00 and 1.10 9 per cubic centimeter are preferred.

The silicate glass coating of the polymer "precursor" is preferred produced by radio frequency atomization.

(The term "silica glass" as used herein refers to it on a glass that contains silicon oxides with or without other metal oxides.) A magnetron radio frequency nebulizer unit is best used. So lets a uniform coating around the spherical outer surface of the "precursor" without achieve overheating of the polymer substrate. A coating thickness in the range of 300 Å to 1 / µm has virtually no measurable effect on the overall density and is suitable. A suitable magnetron sputtering device is available from Sloan Manufacturing Company manufactured and sold as Model No. S301. Another coating process, which can be used is chemical vapor deposition.

The polymer beads are placed in a flat aluminum tray or housed in a container attached to a platform within the atomization system is attached.

Preferably, the beads are introduced through a vacuum lock, so that the pumping time is kept short. The atomization system consists of one Vacuum chamber in which the atomization source is housed and which is on a Pressure less than or equal to 3x10 6 must be evacuated before proceeding with the deposition can be started. Higher pressures result in poor quality films that is mainly expressed in the fact that the Coating trays the steam treatment Cannot withstand in the autoclave. After reaching the required base pressure, the system with the atomizing gas (argon) up to a pressure of about 10 m Torr filled. When using the Sloan Model S310 atomization system, the flat container that holds the beads, 7.5 to 10 cm in diameter. Before and during the deposition, the platform to which the shell is attached is made vibrates in the horizontal direction. Jump through the vibration of the platform the polymer spheres randomly back and forth and rotate so that they are uniform be coated.

In general, a uniform coating is necessary when a thinner one Coating is supposed to survive the steam treatment in the autoclave. It turned out that the uniformity of the coating in the case of beads of a certain size depends on the frequency of vibration and amplitude depends.

For polymer beads with diameters in the range between 100 µm and 200 µm, the optimum vibration frequency and amplitude was 37Hz at approximately 1.5 mm.

To achieve maximum throughput, the deposition rate should can be maximized when atomizing. The maximum rate of deposition that can be achieved depends mainly on the maximum temperature to which the substrate can be heated without affecting the surfaces of the polymer beads be affected. It has been found that a speed of 0.05 / µm per hour on a smooth coating of a Pyrex or silica target Polystyrene beads with diameters of approximately 150 µm. With the one described and special atomization system, this speed resulted at an argon pressure of 10m Torr at an operating power of 0.75 kW. To achieve of complete surface coverage is a minimum coating thickness of approximately 0.03 / µm is necessary. So that the glass-coated polymer beads can be reused they have to be washed again and treated again in the autoclave can. It has been found that a tensile thickness of at least 0.1 µm survived two wash and autoclave cycles and continued active growth of KB cells, MRC-5 cells, Walker 256 carcino-sarcoma cells, and murine fibrosarcoma cells are supported.

The microcarriers described above have quite a number of significant ones Benefits on. The main one is that they are made much cheaper can be considered as the above-mentioned microcarriers made entirely of glass, but that they still have the advantages of the glass surface in the Inherent cell anchorage. They also have an overall density that is found in the culture medium is suspendable, i.e. a neutral buoyancy in the respective medium. Because of The preferred radiofrequency sputtering process also allows the glass composition vary slightly.

However, tall silicate or pure silicate glasses are becoming the present day envisaged for most cell growth applications. The spherical ones Leave polymer substrates at the base density of approximately 1 g per cubic centimeter Of course, they can be produced very inexpensively in different sizes (or to buy).

Another important advantage is that there is an intermediate layer of magnetic material, e.g. a nickel coating, on the polymer substrate can be deposited prior to application of the outer silicate glass layer.

This is preferably done, as described above, in a radio frequency atomization process. The magnetic Layer has no significant influence on the Microcarrier density. The microcarriers can easily be taken out of suspension in one Culture medium can be removed, for example, by placing a permanent magnet in the medium is introduced. The magnetic material can also be used in other ways in the microcarrier, for example by inclusion within the polymer substrate.

Claims (7)

PATENT CLAIMS: 1. Microcarriers for use as growth sites for anchorage-dependent cells in an aqueous cell culture medium, characterized in that that it is a spherical substrate of polymer material with a density of approximately 1 g per cubic centimeter and a thin, continuous surface coating Includes silica glass that completely surrounds the substrate. 2. Microcarrier according to claim 1, characterized in that the diameter of the substrate in the range between 50 / µm and 500 / µm and the density in the range between 1.00 g per cubic centimeter and 1.10 g per cubic centimeter and that is the thickness of the coating is in the range between 300 Å and 1 / µm. 3. Microcarrier according to claim 1 or 2, characterized in that it comprises a second coating of magnetic material which is located below the Silicate glass coating surrounds the substrate. 4. The method for producing a microcarrier according to claim 1, characterized by the following steps: a) It becomes a spherical "precursor" of polymer material chosen at a density of about 1 gram per cubic centimeter; b) the "forerunner" is covered with a layer of silicate glass. 5. The method according to claim 4, characterized in that the step b is carried out in a radio frequency nebulization process. 6. The method according to claim 4 or 5, characterized in that before the following additional step is included in step b: c) the "precursor" is covered with a layer of magnetic material, the layer being made of Silicate glass is placed over this layer of magnetic material. 7. The method according to claim 6, characterized in that the step c is carried out in a radio frequency atomization process.