WO2020177789A1

WO2020177789A1 - Cell culture support for bioreactors

Info

Publication number: WO2020177789A1
Application number: PCT/DE2019/000325
Authority: WO
Inventors: Hans Hoffmeister
Original assignee: Zellwerk Gmbh
Priority date: 2019-03-06
Filing date: 2019-12-13
Publication date: 2020-09-10

Abstract

The invention relates to a cell culture support for bioreactors for insulating, propagating and harvesting cells, in particular NK-cells, T lymphocytes and tumour-infiltrating lymphocytes (TIL) and mesenchymal stem cells from bone marrow, wherein the support comprises a specific covering for the respective type of cell (adhesive; receptor/ligand; co-receptor etc.).

Description

Cell culture carriers for bioreactors

The invention relates to a cell culture carrier for bioreactors for isolation,

Multiplication and harvesting of cells, in particular of NK cells, T lymphocytes and tumor infiltrating lymphocytes (TIL) and mesenchymal stem cells from bone marrow, the carrier being specific for the respective cell type

Has coating (adhesive; receptor / ligand; co-receptor, etc.).

A large number of cell culture carriers for culturing and multiplying cells are known from the prior art. For the cultivation of cells in high density it is necessary that they adhere to an artificial or natural substrate or come into contact with the surface of the cell culture carrier in order to ensure the survival, the multiplication of the cells and / or the preferential growth of a contained phenotype . The production of a desired cell type primarily requires that the substrate surface enables a particular / specific prominent cell adhesion or cell contact and has the ability to support cell growth.

DE 42 06 585 A1 discloses a device for treating cell cultures, in particular hepatocytes, on plate-like cell culture carriers, with at least some of the surfaces of the cell culture carriers being gas-permeable. On the

A collagen layer is applied to cell culture carriers, on which or in which the cell culture is arranged. The next cell culture carrier is arranged a short distance above the collagen layer. Culture medium can be introduced into the space between the collagen layer and the next cell culture carrier.

DE 69 022 778 T2 states that for the adhesion to cells

High molecular weight materials are required, their surfaces with

Low temperature plasma, sputtering, UV light, electron beams are treated and which are covered with a monomolecular, adhesive film that is covered with

Contains cell adhesion factors and cell growth factors treated materials. Cell-adhesive films made from collagen, fibronectin, vitronectin and laminin are known.

The invention according to DE 69 115 928 T2 relates to a support for the cultivation of adhesion-dependent cells (CAD) in the form of microcarriers made from a

biocompatible material. According to the invention, a support for cultivation is provided provided by CAD in the form of microcarriers made of a biocompatible material, the microcarriers having a two-dimensional geometry and having two adjacent surfaces for adhesion with a thickness of 35 µm or less. The cells can adhere and develop on either of the two surfaces without the cells being able to penetrate between the two surfaces. The microcarriers consist of a polymer material, for example a polymer with numerous aromatic groups and especially polystyrene, but also discs made of hydrophilic material such as cellophane or

Poly-D-hydroxybutyrate. The microcarriers are biocompatible, with

Biocompatibility is understood that the CAD on the surfaces of the

microcarriers according to the invention adhere and can multiply there. For this purpose it is provided that the two surfaces of the microcarriers according to the invention are coated with a thin metal layer. This coating has a titanium base, but platinum is also considered

DE 10 2004 053 164 A1 relates to coated microcarriers for the cultivation of animal and human cells, mainly in cells filled with nutrient medium

mixed vessels, such as fixed bed or fluidized bed reactors. The microcarrier consists of a particulate, inorganic, porous or non-porous matrix material, which is solidified into one piece, and a cell culture-compatible, hydrolysis-stable epoxy-amine-polymer coating, which is deposited on this piece. The size of the particles is in a range from 100 to 1000 µm, preferably in a range from 180 to 590 µm. Suitable

Matrix materials for the microcarriers are, for example, glass, ceramics, silica gel, gypsum, metal oxides, metal silicates or tricalcium phosphates. A glass from the Si02-Ca0-Na20-Mg0-K20-AI203-Fe203 system is particularly advantageous as a carrier material. The crosslinked epoxy-amine polymer used as a coating for the inorganic matrix material is produced from one or more epoxy components and one or more amine components.

In DE10 2005 035434 A1 cell culture carriers are disclosed on which cells adhere and grow efficiently and from which the grown cells are simply detached. The cell culture carriers each contain a granular base material and a cover layer that covers the surface of the base material. The top layer consists mainly of a calcium phosphate-based apatite, in which part of the calcium is missing. Such cell culture carriers are used in cell cultures in where the cells adhere to the surfaces of the cell culture carriers and grow there. In particular, these cell culture carriers are used in three-dimensional, high-density cultures (suspension cultures). The Ca deficiency in the calcium phosphate-based apatite is preferably in a range of 1 to 30 mol%.

The invention according to WO 2010/136136 A3 relates to a method for producing a coated cell culture carrier for the in vitro cultivation of stem cells, in particular for the cultivation of mesenchymal stem cells, wherein a

Solution, which comprises a polyurethane urea, is applied to a cell carrier and dried. The polyurethane urea is previously by converting at least one polycarbonate polyol component, at least one

Polyisocyanate component and at least one diamino component produced. Antioxidants or pigments, but also grip aids, dyes,

Matting agents, UV stabilizers, light stabilizers, water repellants, water repellants and / or flow aids can be used as additives. A wide variety of substrates such as glass, silicon wafers, metals, ceramics and plastics are coated. The coating of cell culture carriers made of glass, silicon wafers, plastic or metals is preferred. The metals mentioned are, for example, medical stainless steel and nickel-titanium alloys. As

The polymer materials from which the cell culture carrier is constructed are polyamide; Polystyrene; Polycarbonate; Polyether; Polyester; Polyvinyl acetate; natural and synthetic rubbers; Block copolymers of styrene and unsaturated

Compounds such as ethylene, butylene and isoprene; Polyethylene or copolymers of polyethylene and polypropylene; Silicone; Polyvinyl chloride (PVC) and polyurethanes are used. For better adhesion of the polyurethanes according to the invention to the cell culture carrier, this can be used as a substrate before application

Coating materials still further suitable coatings are applied. The coating of glass or silicon wafers for the production of cell culture carriers is particularly preferred. As a solvent for the production of the

Polyurethane urea solutions are all conceivable solvents and solvent mixtures such as dimethylformamide, N-methylacetamide, tetramethylurea, N-methylpyrrolidone, aromatic solvents such as toluene, linear and cyclic esters, ethers, ketones and alcohols. Examples of esters and ketones are

Ethyl acetate, butyl acetate, acetone, v-butyrolactone, methyl ethyl ketone and

Methyl isobutyl ketone is used. Mixtures of alcohols with are preferred Toluene. Examples of alcohols that can be used together with toluene are ethanol, n-propanol, iso-propanol and 1-methoxy-2-propanol. The

Coatings from the polyurethane urea solutions can by means of

different methods can be applied to the cell culture carrier. Suitable coating techniques are, for example, knife coating, printing, transfer coating, spraying, spin coating or dipping.

In WO 2016/202329 A1 a bioactive coating material for

Coating of plastic materials for cell cultures, preferably

Disposable plastic materials, for example made of polystyrene (TCP) disclosed as plastic, in particular a cell-instructive coating material for

Cell culture materials made of plastic, for example polystyrene. The bioactive one

Coating material comprises a polymer conjugate each of a polymeric anchor molecule with surface-active anchor groups on the one hand and of one or more biologically active molecules on the other hand, the anchor molecule being an amphiphilic molecule with a hydrophobic part made of styrene, methacrylic acid or isobutene units and a hydrophilic part made of maleic anhydride or Is maleic acid units.

EP 1 270 533 A2 describes a carrier material for cell culture carriers made of ceramic materials for improved support for the culture of anchorage-dependent eukaryotic cells and prokaryotic cells which comprise a crystalline core oxide ceramic which is dispersed with an amorphous polyanionic intergranular phase. The ceramic material consists of an oxide of the crystalline core oxide ceramic, preferably from the group consisting of zirconium (IV) oxide, aluminum oxide, silicon (IV) oxide, titanium (IV) oxide,

Hafnium (IV) oxide, cerium (IV) oxide and mixtures thereof. The

polyanionic compound in the amorphous intergranular phase is selected from the group consisting of phosphates, manganates, molybdates, tungstates or mixtures thereof.

With the cell culture carrier shown in the prior art from a

Carrier material with a coating of cell adhesive material, cells can only be isolated, multiplied and harvested in an amount that is unsuitable for cell therapy, the cells also having a low cytotoxic effect. The object of the invention is to develop a cell culture carrier with the aid of which cells, in particular NK cells and autologous, tumor-infiltrating lymphocytes and autologous T lymphocytes, but also mesenchymal stem cells, are extracted

Bone marrow can be isolated in sufficient quantities, specifically activated ex vivo, and multiplied and harvested. The harvested immune cell cells are used as a suspension of living cells in vivo for a therapy against tumor cells, the cells having a high specific toxicity against the unique mixture of mutated lymphocytes of a patient's individual tumor or its metastases. Lymphocytes each individual tumor cell

The object is achieved by a cell culture carrier (20) which consists of a hydrophilic carrier body (21) treated with low-temperature plasma with a cell-adhesive coating (22), with activating antibodies being incorporated into the cell-adhesive coating (22). The carrier body (21) consists of plastic, glass, metal or ceramic materials. The cell-adhesive coating (22) consists of fibronectin, retro-nectin, collagen, gelatine, laminin, quinitine and / or

Combinations of these. Immunoglobulins, ß-lymphocytes, anti CD 16, anti CD3, anti CD28, anti137, anti CD 39, anti CD103 and / or combinations thereof are incorporated into the cell adhesive coating as activating antibodies.

In one version of the invention, the cell-adhesive coating (22) contains the antibody 4-1 bb in connection with the cytokines as activating antibodies

Interleukin 12 or a mixture of Interleukin 12 and Interleukin 2 incorporated.

The cell culture carrier according to the invention can be used in fixed bed bioreactors, fluidized bed reactors, roller bioreactors, hollow fiber bioreactors or meander bioreactors, which are used with a cell adhesive or coating with activating antibodies contained therein for isolating and multiplying cells. With the cell culture carrier according to the invention it is also possible to

small cut pieces of tissue to allow certain cells to grow into the medium in the bioreactor as well as the cells at the same time

to expand, differentiate, activate, stimulate dynamically and stress-free and then to separate from tissue remnants. In particular, NK cells, tumor-infiltrating autologous T lymphocytes (TIL) from tumor tissue,

Metastatic tissue, its surrounding tissue and lymph nodes that are infected by tumor cells, but also the total fraction or individual cell types the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, immune cells therein, are obtained, the bioreactor working in perfusion mode.

The invention will now be explained in more detail using examples, with FIG. 1 being a sketch of the cell culture carrier (20) and FIG. 2 being a sketch of the meandering bioreactor from DE 20 2018 004 857.7 with the top (18) of the bottom surface (6) as Cell culture carrier (20) shows where

1 bioreactor vessel

2 lids

3 underlay chamber

4 mandible perfusion chamber

5 Overiay Chamber

6 Base plate with coating (22) as a cell culture carrier (20)

7 slide

8 partition

9 Infeed underlay atmosphere

10 Discharge of the Unterlay atmosphere

11 Supply of culture medium

12 Removal of culture medium

13 Infeed overlay atmosphere

14 Dissipation of overlay atmosphere

15 outlet spouts

16 mesh

17 Overflow for cell broth

18 Top of the base plate (6)

19 channels 20 cell culture carriers

21 carrier body

22 coating mean.

Embodiment 1

The meander perfusion bioreactor used in the exemplary embodiment is described in DE 20 2018 004 857.7 by the same applicant and consists of a rectangular bioreactor vessel (1) made of preferably clear polymer material, which is sterile closed with a lid (2). The bioreactor vessel (1} is divided into three chambers (3, 4, 5) arranged one above the other, the lowest chamber

(3) as an underlay chamber, the chamber arranged above the underlay chamber (3)

(4) are designed as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) as an overlay chamber. The underlay chamber (3) and the meander perfusion chamber (4) are through a perforated base plate (6) with a bottom on the perforated base plate (6)

arranged semipermeabten film (7) separated from each other. On the top (18) of the base plate (6) are strip-shaped dividing and one

Meander-shaped flow through the meander perfusion chamber (4) with gas and medium enforcing partition walls (8), the distance (A) of the partition walls (8) from each other and from the side and end walls of the meander perfusion chamber (4) is chosen so that an averaged laminar overflow with a Froude number <0.005 is formed in the flow filament in the channel formed by the partition walls (8). This distance (A) is 2 to 25 mm, preferably 4 to 6 mm. The bottom plate (6) of the meander perfusion chamber (4) is as

Carrier body (21) of a cell culture carrier (20) and is provided with a cell-adhesive coating (22) on the upper side (18) pointing in the direction of the meander perfusion chamber (4). For this purpose, fibronectin dissolved in water mixed with anti-CD3 as an activating antibody is brought into the channel (19) of the meander bioreactor formed by the partition walls (8), distributed evenly by tilting the meander bioreactor and dried out. At the underlay chamber (3) are inlets and outlets (13, 14) at different heights for a regulated flow of gases, in particular air, mixed with different volume percentages of oxygen (5% to 50%, preferably 10 to 25% volume percent). The oxygen then reaches the meander perfusion chamber (4) by diffusion through the semipermeable film (7) and through the perforated base plate (6), where the culture medium is then transferred to the

Flow through the meander perfusion chamber (4) is supplied with O2 by perfusion. The meander perfusion chamber (4) is provided with inlets and outlets for culture media (11, 12), the culture medium consisting of a CellGrow medium , which is temporary with a specific mixture of AB human serum, cytokines, antibodies and irradiated human feeder cells

is supplemented. The autologous tumor tissue pieces in a size of 1 to 2 mm ³ , which were removed and comminuted from the patient during an operation, are added to this culture medium and fed with the culture medium into the meander perfusion chamber (4). The comminuted pieces of tissue are evenly distributed in the bioreactor and cultivated in the perfusion operation, whereby the

Culture medium via which the overlay chamber (5) is supplied with the appropriate overlay atmosphere in a regulated manner via the inlets and outlets (13, 14).

In a cultivation run over 7 to 14 days, TILs normally grow out of the tissue pieces in large quantities. The TILs are separated from the tissue residues as filtrate via the outlet connection (15) of the overlay chamber (5) of the bioreactor vessel (1) provided with sieve fabric (16).

As the number of cells increases in the course of the increase in the number of cells (TIL or other cells), the supply of fresh medium is continuously increased, the increased supply of fresh medium being automatically controlled by a corresponding algorithm. The bottom flow remains close to one

Froude number 0. This slow movement of the culture medium avoids whirling up the cells. However, there is frequent contact between the components of the cell-adhesive coating and the cells that have sedimented on it.

The flow conditions described prevent cell stress and ensure a homogeneous supply of nutrients over the entire cell layer growing at the bottom of the channel. Embodiment 2;

In a meander bioreactor, as described in Example 1, with the cell culture carrier (20) formed as the bottom surface (6) of the channel (19) of the meander bioreactor from the carrier body (21) and a cell-adhesive coating (22) with anti - CD3 as an activating antibody, TILs are obtained from shredded pieces of tissue, whereby the conditions of the floor flow in the meander perfusion chamber (4) close to a Froude number 0 are maintained here as well. The starting material for this are pieces of tissue that arise during the surgical removal of a tumor, tumor metastases or lymph nodes affected by tumor cells. The comminuted pieces of tissue are filled in through the larger-sized stopper that closes in a sterile manner and distributed evenly on the floor by swiveling the medium-filled meander perfusion bioreactor. After closing the filler neck, cultivation is started as in Example 1. After 3 to 7 days, the tumor-infiltrated T lymphocytes (TIL) contained in the tumorous tissue pieces grow out in large quantities. Depending on the medium chosen and the supplements added, the TIL in this

Process phase activated, through further contact with the initially still existing mixture of mutated tumor cells from an individual tumor piece (obtained from a tumor resectate), mutated tumor cells are specifically primed. As a rule, 1.5 to 2 × 10 ⁹ TILs grow within a week in the meander perfusion bioreactor with a floor area of 30 cm ² . After shaking up the meander perfusion bioreactor, the TIL are given through the side outlet port (15), which is provided with a sieve mesh, into a sterile vessel or into a larger meander bioreactor for further expansion. The

Pieces of tissue from which the TIL has grown are completely retained by the sieve mesh of the first meander bioreactor.

Embodiment 3

In a meander bioreactor, as described in Example 1, the base plate (6) of the meander perfusion chamber (4) is designed as a support body (21) of a cell culture carrier (20) and is on the in the direction of the meander perfusion chamber (4) facing top (18) provided with a cell-adhesive coating (22). For this purpose, fibronectin dissolved in water is mixed with the antibody 4-1 bb as activating antibody in conjunction with the cytokine IL12 or a mixture of IL 2 and IL 12 in the channel (19) formed by the partition walls (8) of the meander bioreactor, evenly distributed by tilting the meander bioreactor and dried out. Thereafter, TI Ls are obtained from comminuted pieces of tissue, as described in exemplary embodiments 1 and 2, the conditions of the bottom flow in the meander perfusion chamber (4) close to a Froude number 0 being maintained here as well.

Claims

1. Cell culture carrier (20) for bioreactors for isolating and multiplying cells consisting of a hydrophilic carrier body (21) treated with low-temperature plasma and having a cell-adhesive coating (22), characterized in that activating antibodies are incorporated into the cell-adhesive coating (22) .

2. cell culture carrier (20) according to claim 1, characterized in that the activating antibodies are immunoglobulins, ß-lymphocytes, anti CD 16, anti CD3, anti CD28, anti 37, anti CD39, anti CD103 and / or from

Combinations thereof are incorporated into the cell-adhesive coating (22).

3. Cell culture carrier (20) characterized in that as activating

Antibodies of the antibodies 4-1 bb in combination with the cykins Interleukin 12 or with a mixture of Interleukin 12 and Interleukin 2 is incorporated into the cell-adhesive coating.

4. Cell culture carrier (20) according to claim 1, characterized in that the cell-adhesive coating (22) consists of flbronectin, RetroNectin, collagen, gelatine, laminin, quinitine and / or combinations thereof.

5. cell culture carrier (20) according to claim 1, characterized in that the carrier body (21) made of plastic, glass, metal or ceramic

Materials.

6. cell culture carrier (20) according to claim 4, characterized in that the carrier body (21) consists of a clear polymer material.

7. cell culture carrier (20) according to claim 1 to 5, characterized in that the cell culture carrier (20) in bioreactors, fixed bed bioreactors, fluidized bed reactors, roller bioreactors, hollow fiber bioreactors or meander Bioreactors or as a part of these are used for the isolation and multiplication of cells.