WO2020078498A1

WO2020078498A1 - Meandering perfusion bioreactor for the differentiation, activation, stimulation and separation of cells

Info

Publication number: WO2020078498A1
Application number: PCT/DE2019/000269
Authority: WO
Inventors: Hans Hoffmeister
Original assignee: Zellwerk Gmbh
Priority date: 2018-10-20
Filing date: 2019-10-11
Publication date: 2020-04-23
Also published as: DE202018004857U1

Abstract

The invention relates to a novel kind of bioreactor, by means of which, in a single closed method step, it is possible both to leave cells provided from small pieces of cut tissue to grow in the medium situated in the bioreactor and simultaneously also to activate the cells, multiply them, and then separate them from tissue residues. In particular, tumour-infiltrating autologous T-lymphocytes (what are known as TIL) can in this way be obtained from tumour tissue, metastasis tissue, tissue surrounding same, and from lymph nodes affected by tumour cells. The directed laminar flow of medium over the cells leads to a rapid growth of the cells. In the case of TIL, in particular the cells that have specific cytotoxicity in relation to the tumour cells from which or from the surroundings of which they were obtained are preferably expanded. A technique that integrates a plurality of method steps is not known from the prior art. The prior art also does not describe a bioreactor by means of which cells and in particular TIL can be produced safely in sterile conditions and in therapeutically useful amounts on the basis of their high cytotoxicity.

Description

Meander perfusion bioreactor for the differentiation, activation, stimulation and separation of cells

The invention relates to a meander bioreactor, in which it succeeds

to cut small pieces of tissue out of certain cells both in the medium in the bioreactor and the cells at the same time

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

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

DE 695 34 132 T2 describes a cell culture device which comprises a container with a cell culture compartment, the container from a lower one

gas permeable layer and an upper sheet that is selectively permeable to compounds of a given size and impermeable to cells. The container is designed to hold a culture medium between the upper leaf and the lower gas permeable layer.

The container consists of

a basal medium compartment, at least a portion of which is above the upper sheet, a basal medium may be on the upper sheet;

only a single access opening to the cell culture compartment;

- an access opening to the basal medium compartment;

and

a gas-permeable substrate which is arranged below the gas-permeable layer and is partially in contact therewith, whereby the majority of the gas-permeable layer is held in a substantially horizontal position, so that cells of a suspension are distributed along the horizontal section of the gas-permeable layer can and a gas exchange with the

Cell culture compartment is not significantly hindered. DE 693 32 374 T2 describes a bioreactor for cultivating human cells and / or tissues, including human stem cells or human

described hematopoietic cells, which consist of a housing that a

Defined cell culture chamber into which human cells and / or tissues, including human stem cells or human hematopoietic cells, are introduced and in which they can be cultivated, wherein this cell culture chamber is designed such that it contains a multiplicity of inlet openings substantially equidistant to a multiplicity of outlet openings so that cell culture medium can flow through. The bioreactor continues to write

• Means for perfusing a liquid cell culture medium through the

Cell culture chamber;

• means for contacting the liquid cell culture medium perfused through the cell culture chamber with an oxygen source so that the liquid cell culture medium perfused through the cell culture chamber is enriched with oxygen, these means for contacting contain a gas permeable and liquid impermeable membrane; and

• Means for continuously, periodically or discontinuously harvesting non-adherent cells from the cell culture chamber by the flow of the

Culture medium.

The gas permeable and liquid impermeable membrane is built into the housing so that the housing is divided into the cell culture chamber on one side of the membrane and a gas chamber on the other side of the membrane. The bioreactor includes means for perfusing respiratory gases through this cell culture, which includes means for supplying the respiratory gases to the gas chamber, whereby the respiratory gases flow through the membrane and thereby through the membrane

Cell culture chamber pass through, and means for withdrawing used

Breathing gases from the gas chamber.

The invention according to DE 69614535 T2 relates generally to a device for storing and cultivating biological cells ex vivo and in particular to a device of this type which stores the cells in a portable cassette and breeds while maintaining a sterile system that is isolated from the outside environment

The device for ex vivo storage and cultivation of biological cells comprises a portable cell growth cassette which includes

• a housing,

A cell growth chamber within the housing configured to carry an amount of biological cells and a growth medium, the cell growth chamber having a medium inlet opening and a medium outlet opening,

• a medium container that is connected to the medium inlet opening of the

Cell growth chamber is connected and configured to contain a growth medium, and

• a waste container that is connected to the medium outlet opening of the

• Cell growth chamber is connected and configured by the

• growth chamber drained medium

• Record, with the cell growth chamber, the medium container and

• the waste container is a sterile one sealed off from the outside environment

• form a system;

The cell growth chamber contains a gas-permeable / liquid-impermeable membrane which defines a part of the cell growth chamber and a gas chamber which is located on the side of the membrane opposite the cell growth chamber. The portable cell growth cassette still includes one

A gas supply port supported by a rear wall of the housing and a gas supply line connecting the gas supply port to the gas chamber so as to supply gas to the gas chamber for permeation through the membrane to the cell growth chamber.

The invention according to CN 1699558 relates to an industrial one

Cultivation technology for killer cells to induce cell factor for

hematopoietic stem cells using a bioreactor comprising obtaining umbilical cord blood hemapoietic stem cells, inducing the hematopoietic stem cells in DC cells with cytokine, completing the function of tumor antigen to present the selected activation of killer T lymphocytes with specific killing effects on tumors

complete linking the preparation of the gelose microcarrier for the monoclonal antibody (CD3McAb), the activation of the CIK cells and the continuous cultivation of CIK cells in the bioreactor.

The invention according to DE 10 2011 106 914 B4 / EP25 43 719 A1 discloses a Mänder bioreactor for dynamic and stress-free expansion,

Differentiation and harvest of the total fraction of hematopoietic cells,

Stem cells, progenitor cells, but also of isolated, stimulated, activated fractions of these cells, apart from embryonic cells, the bioreactor working in perfusion mode. The bioreactor consists of a bioreactor vessel, the bottom surface of which is provided with staggered partition walls, which result in a meandering guidance of the media with a laminar flow. The meander bioreactor can be connected in series with other meander bioreactors in a modular manner via shut-off valves. After being fed into the bioreactor vessel, the cells form a coherent cell layer which almost covers the bottom surface of the meander bioreactor, the cells often being in close contact with one another. The cell clumps which arise during the expansion are dissolved by shaking and harvested or become a further module-like bioreactor with a larger bottom surface of the bioreactor vessel corresponding to the expansion rate of the previous bioreactor

Expansion fed.

In our own invention according to DE 10 2018 000 561.6, a meander bioreactor and a two-stage process for the ex vivo extraction of cells are described, which in the first stage of the process grow out of shredded tissue parts into the medium in the bioreactor vessel (1), be activated and increased simultaneously and after reaching a certain density

adult and expanded cells are separated from the tissue parts. The starting tissues, which are affected by tumor cells, preferably originate from organ tumor or metastatic operations or the removal of their immediate surrounding tissues or from lymph nodes. The

The first stage is carried out in a closed meandering Bioreactor vessel (1) which is perfused in a directional manner with a conventional medium and the medium is temporarily supplemented with a mixture of AB human serum, cytokines, antibodies and irradiated human feeder cells. The

small pieces of tissue are particularly suitable for

Obtaining tumor-infiltrated T-lymphocytes (TIL), but also for tumor-infiltrated natural killer cells (TINK). The meander bioreactor consists of one

Rectangular bioreactor vessel made of preferably clear polymer material, which is sterile closed with a lid, the bioreactor vessel in three

chambers arranged one above the other is divided and the lowermost chamber as a sublay chamber, the chamber arranged above the sublay chamber as

The meander bioreactor chamber and the chamber arranged above the meander bioreactor chamber are designed as an overlay chamber and the underlay chamber and the meander bioreactor chamber are separated from one another by a perforated base plate with a semipermeable film arranged at the bottom of the perforated base plate are.

The invention has for its object to develop a meandering perfusion bioreactor for the production of individual cell types of the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, immune cells and NK cells therein for therapeutic applications and transplantation purposes, which enables sufficient amounts of autologous tumor infiltrating T lymphocytes (TIL), but also other immune cells, e.g. tumor infiltrating NZ cells (TINK) from tumor tissue, metastasis tissue, their surrounding tissue and from

Lymph nodes that are affected by tumor cells, from small-cut pieces of tissue (those from surgical tissue parts or from biopsies

grow out initially in a closed perfusion process, multiply continuously, are activated in the process and can be harvested after reaching a certain cell density

The bioreactor should have a simple structure and be able to be manufactured as a disposable version. The procedures for cell expansion, differentiation, stimulation and harvesting should be carried out safely, easily and transparently in order to meet all requirements for

Cell preparations with regard to GMP conformity and obtaining

Manufacturing permits for the hematopoietic manufactured in the bioreactor

To fulfill cell preparations. Furthermore, the bioreactor should be easy to use in everyday clinical operation. According to the invention, the object is achieved by a meander perfusion bioreactor for the ex vivo extraction of cells, which 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

Sublay chamber, the chamber (4) arranged above the sublay chamber (3) as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) are designed as an overlay chamber and the sublay - Chamber (3) and the meander perfusion chamber (4) through a perforated

Base plate (6) with a semi-permeable film (7) arranged at the bottom of the perforated base plate (6) are separated from one another. On the top (8) of the base plate (6) are strip-shaped dividing and one meandering

Flow through the meandering perfusion chamber (4) with gas and medium-enforcing partition walls (8). Surprisingly, it was found that the bottom surface (18) of the bottom plate (6) of the meander perfusion chamber (4, the width and the height of the channels (19) formed by the partition walls (8) force contact between the seeding cells, in Depending on the amount of cells to be grown and depending on the amount of seed cells of the starting amount have freely selectable size.

In one embodiment of the invention, the base surface (18) of the base plate (6) of the meander perfusion chamber (4) is between 20 to 1500 cm ² , the height of the base plate (6) of the meander perfusion chamber (4) arranged channels (19) between 1, 8 to 6 cm and the distance (A) of the partitions (8) of the channels (19) from each other and from the side and end walls of the meander perfusion chamber (4) between 2 to 25 mm freely selectable.

In a further embodiment of the invention, the bottom surface (18) of the

Bottom plate (6) of the meander perfusion chamber (4) between 30 to 500 cm ² and the height of the channels (19) formed by the partitions (8) between 1, 8 cm and 2.5 cm designed freely.

In a still further embodiment of the invention, the distance (A) of the partitions (8) of the channels (19) from one another and from the side and

The end walls of the meander perfusion chamber (4) can be freely selected between 4 and 6 mm. Due to the design of the bottom surface (18) and the height and width of the channel (19), an average laminar upstream flow with a Froude number <0.005 and a bottom flow with a Froude number close to 0 occur in the stream filament of the channel without whirling up the cells.

The meander principle only makes it homogeneous and gradient-free

Supply of cells in this perfusion bioreactor is possible, under selectable flow rates, variable supply of fresh medium to circulating medium, hypoxemic, normoxemic or hyperoxemic in sublay / overlay chambers and selectable area and volume ratios of the perfusion chamber (4).

The lower layer chamber (3) is provided with inlets and outlets (9, 10) at different heights for the flow of gases, the meander perfusion chamber (4) with inlets and outlets for culture media (11, 12) and provide the overlay chamber (5) with inlets and outlets (13, 14) for overlay gases and with an outlet connection (15), on which a screen fabric (16) is arranged in the interior of the overlay chamber (5) before the outlet , wherein an arranged overflow (17) for the used cell broth is arranged adjustable in height on the feed (11) of the meander perfusion chamber (4).

In one configuration of the invention, the meander perfusion bioreactor can be connected in a modular manner to a plurality of meander perfusion bioreactors via the overflow (17) for the cell broth (with shut-off devices not shown in more detail).

The process for the ex vivo recovery of cells consists of two stages, the cells in the first stage of the process growing out of shredded tissue parts into the medium in the bioreactor vessel (1), being activated and multiplied at the same time and after reaching a certain density adult and expanded cells are separated from the tissue parts. The starting tissues, which are affected by tumor cells, preferably originate from organ tumor or metastasis operations or the removal of their immediate surrounding tissues or also from lymph nodes. The process in the first stage takes place in a closed meander perfusion bioreactor vessel (1), which is perfused in a directional manner with a customary medium and the medium is temporarily supplemented with a mixture of AB human serum, cytokines, antibodies and irradiated human feeder cells. The small pieces of tissue are particularly suitable for the extraction of tumor-infiltrated T-lymphocytes (TIL), but also for tumor-infiltrated natural killer cells (TINK)

After 7 to 14 days of expansion, the fully grown and propagated TIL are placed over the outlet connection (15) of the

The bioreactor vessel (1) is filtered off from the tissue residues and harvested in pure form. The TIL separated in this way are centrifuged and placed in freezing medium in vials

resuspended, aliquoted and cryopreserved.

With appropriately supplemented media, tumor-infiltrated NK cells (TINK) and other cell types can also be cultivated in the meander bioreactor.

For clinical applications, cryopreserved vials are thawed in the second stage in such a way that therapeutically useful amounts of TIL are produced on schedule in a second expansion phase. For this purpose, after washing out the freezing medium, the TIL are suspended again in a suitable medium. The TIL suspension (50 to 100 million vital cells) is transferred to another meander bioreactor installed ready for operation. This second process stage for the further expansion of TIL is carried out in a bioreactor which is similar to the meandering bioreactor described for the first stage, but which is a larger one

Settlement area in the ratio of 3 to 1 to 20 to 1. Freshly supplemented medium is used for further propagation.

After 12 to 20 days of expansion, the meander perfusion bioreactor of the second step regularly grows more than 500 million TIL. The TIL are harvested, washed and resuspended in NaCI solution with human albumin. The TIL suspension is filled into an infusion bag. Viability and cell count are analyzed on the day of the cell harvest according to the pharmacopoeia sterility, marker profile and paracrine production of the cells. A maximum of 100 ml of NaCI solution with human albumin is used for 500 million TIL. The TIL suspension is at

Maintained at room temperature and immediately transported to the clinical partner. TIL must be applied after 12 to 24 hours at the latest.

Excess amounts of TIL are cryopreserved. They are available for the subsequent production of additional cans of TIL

In an interpretation of the method, the increased TIL can be compared with the

Filter fabric (16) provided outlet connection (15) of the bioreactor vessel (1) from the tissue residues and filter the cell suspension with the TIL directly into the second stage in a meandering bioreactor of the same type as in the first stage but with transferred to a larger settlement area, cultivated for 12 to 20 days, the cells were harvested after the cultivation, washed and resuspended in NaCl solution with human albumin and the cell suspension was filled into infusion bags.

The invention is now based on? explained in more detail using examples, wherein Fig. 1 shows a schematic sectional view of the meandering perfusion bioreactor and Fig. 2 shows a schematic plan view of the meandering perfusion bioreactor and

1 bioreactor vessel

2 lids

3 sublay chamber

4 mander perfusion chamber

5 overlay chamber

6 base plate

7 slide

8 partition

9 Feed underlay atmosphere

10 Exhaustion sublay atmosphere

11 Culture medium feed

12 Removal of culture medium

13 Feed overlay atmosphere

14 Overlay atmosphere discharge

15 outlet spigot

16 sieve fabrics

17 Overflow for cell broth

18 floor space

19 channels

mean.

example 1

The starting material for the production of TIL or TINK and other cells are tissue components which e.g. occur during the surgical removal of solid organ tumors, their metastases or their immediate surrounding tissue or are removed for this purpose. A quantity of tissue with a volume of about 1 ml is usually sufficient as a starting material. The tissue samples are in one

Transport vessel spent with medium. The vessel is closed at

Kept at room temperature and from the place of tissue extraction into the Clean room area for the production of immune cell preparations transported. The surgical tissue pieces are chopped into pieces of 1 to 2 mm ^{3 in} size in a clean room under an LFB and transferred to a meander perfusion bioreactor in the first stage. The shredded pieces of tissue are evenly distributed in the meander perfusion bioreactor and cultivated in the perfusion operation. The meander perfusion bioreactor is ready for operation in a GMP breeder

connected and is continuously monitored by the control unit, largely automatically controlled and documented.

The meander perfusion bioreactor consists of a rectangular one

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 the underlay chamber, which is above the

Lower layer chamber (3) arranged chamber (4) as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) are designed as an overlay chamber.

The underlay chamber (3) and the meander perfusion chamber (4) are separated from one another by a perforated base plate (6) with a film (7) which is tightly fastened thereon and is permeable to oxygen. On the lower layer chamber (3) there are inlets and outlets (9, 10) that allow gases to flow through them.

in particular with mixtures of air and oxygen or with nitrogen and

Enable oxygen in which the proportion of oxygen is regulated. The oxygen diffuses preferably over nitrogen through the gas-permeable film (7), which is tightly attached to the perforated base plate (6) of the meander perfusion chamber (4). When flowing through the meander perfusion chamber (4), regulated amounts of oxygen (by diffusion) get into the cell from both the overlay and the underlay chamber during the expansion of the cells

Culture medium.

The meander perfusion chamber (4) is provided with inlets and outlets for culture media (11, 12), likewise in the overlay chamber (5) inlets and outlets (13, 14) for the overlay atmosphere and a Outlet connector (15) available. A screen fabric (16) is arranged on the outlet nozzle (15) in the interior of the overlay chamber (5) before the outlet. At the outlet (12) of the meander perfusion Chamber (4) is also an overflow (17) for the removal of the spent cell broth. The height of the overflow is adjustable.

The meander perfusion chamber (4) consists of the base plate (6) with strip-shaped arranged on the top (8), dividing the base plate (6) and forcing a meandering flow through the meander perfusion chamber (4) with gases and medium Partition walls (8), the distance (A) of the partition walls (8) from one another and from the side and end walls of the meander perfusion chamber (4) being selected such that the streamline through which the partition walls (8 ) formed channels, forms an average laminar upstream with a Froude number <0.005. With increasing cell number in the course of

Increasing the amount of cells (TIL or other cells) increases the supply of fresh medium, which is automatically controlled by an appropriate algorithm. The bottom flow remains close to one

Froude number 0, which prevents whirling up of the cells. The

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

The overlay chamber (5) has inlets and outlets 13, 14) for the overflow with gases. The same gas / gas mixture as the underlay chamber (5) flows through the overlay chamber (5).

In a cultivation run, cells usually grow out of the pieces of tissue for 7 to 14 days, the adult cells multiply at the same time. In this way, TIL can be produced in large quantities from tumor tissue. But TINK and other cells can also be obtained from tissues in this way. If the

If glucose consumption in the bioreactor reaches 100 to 300 mg per day, the TIL are discharged via the outlet connection (15) provided with sieve fabric (16)

Bioreactor vessel (1) separated from the tissue residues as filtrate.

From the transfer of the tissue pieces in the first stage into the bioreactor vessel (1), the entire process proceeds to the filling of activated and expanded cells in a completely closed vessel system. Cultivation is carried out in a suitable medium which is temporarily supplemented with a specific mixture of AB human serum, cytokines, antibodies and irradiated human feeder cells. Faster

Propagation and activations are occasionally achieved through supplements that are fixed on the surfaces of the bioreactor clot. In the case of TIL and others Immune cells are centrifuged the TIL multiplied and separated in this first stage, resuspended in freezing medium and aliquoted (about 50 million TIL per vial). The samples are stored in the gas phase over liquid nitrogen.

For clinical applications, cryopreserved vials are thawed in such a way that in a second expansion stage, therapeutically meaningful amounts of TIL

be produced on time. For this, after washing out of

Freezing medium the TIL resuspended in suitable medium. The TIL suspension (50 to 100 million vital cells) is transferred to another meander bioreactor installed ready for operation. This second process stage for the further expansion of TIL is carried out in a meander bioreactor which is similar to the meander bioreactor described above, but a larger one

Owns settlement area. Freshly supplemented medium is used for further propagation. After an expansion period of 12 to 20 days, more than 500 million TIL regularly grow up in the meander bioreactor. The TIL are harvested, washed and resuspended in NaCI solution with human albumin. The TIL suspension is filled into an infusion bag. Viability and cell number are determined on the day of the cell harvest and further analyzed according to the pharmacopoeia sterility, marker profile, and paracrine production of the cells. The TIL suspension is kept at room temperature and immediately transported to the clinical partner. TIL must be applied after 12 to 24 hours at the latest.

Excess amounts of TIL are cryopreserved. They are available for the later production of additional doses of TIL. For 500 million TIL, a maximum of 100 ml NaCI solution with human albumin is used.

Example 2:

TIL are obtained from shredded pieces of tissue in a meander bioreactor as described in Example 1. The starting material for this are tissue parts that occur when a tumor is surgically removed, from tumor metastases or from lymph nodes affected by tumor cells. The shredded tissue pieces are filled in through the larger dimensioned and with a sterile sealing stopper and evenly distributed on the floor by swiveling the medium-filled meander perfusion bioreactor. After closing the filler neck, the cultivation is started as in Example 1. After 3 to 7 days, they grow in tumor-infiltrated T-lymphocytes (TIL) contained in the tumorous tissue pieces in quantities. Depending on the medium selected and the supplements added, the TILs are activated in this process phase and specifically primed by further contact with the mutant tumor cells that were still present. They usually multiply to 0.5 to 1.5 x 10 ⁹ TIL. After shaking the meander perfusion bioreactor, the TIL are put through the side outlet nozzle (15), which is provided with a sieve, into a sterile vessel or into a larger meander bioreactor for further expansion. The pieces of tissue from which the TIL have grown are completely retained by the sieve fabric of the first meander bioreactor.

A gradient-free, homogeneous supply to the cells is possible with these meander perfusion bioreactors. In this simple and closed first process step, the meander perfusion bioreactor combines a longer specific priming by the respective individual tumor cells, the activation for faster growth of the TIL and the isolation of a large amount of very pure TIL.

With the meander bioreactor according to the invention and the method according to the invention for the isolation and multiplication of cells from tissue parts of tumors, metastases and other tissues, it is possible in a single, closed process step to grow certain cells from small pieces of tissue into the medium in the bioreactor, as well as activating and multiplying the cells at the same time and then separating them from tissue remnants. In particular, tumor-infiltrating autologous T-lymphocytes (TIL) from tumor tissue, metastatic tissue, their

Surrounding tissue and from lymph nodes that are affected by tumor cells. The directional laminar overflow of the cells with medium in the meander bioreactor leads to rapid growth of the cells. In the case of TIL, the cells, the specific ones, are particularly preferably expanded

Have cytotoxicity to the tumor cells from or around which they are obtained.

In a second process step, a bioreactor which is similar to the meander bioreactor of the first process step but which is a larger one Settled area has produced therapeutically meaningful amounts of TIL for clinical applications.

Claims

1. meander perfusion bioreactor for differentiation, activation,

Stimulation and separation of cells consisting of a rectangular bioreactor vessel (1) made of preferably clear polymer material, which is sterile closed with a lid (2), the bioreactor vessel (1) being divided into three chambers (3, 4, 5) arranged one above the other and the lowest chamber (3) as a sublay chamber, the chamber (4) arranged above the sublay chamber (3) as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) as an overlay - Chamber formed and the underlay chamber (3) and the meander perfusion chamber (4) through a perforated base plate (6) with a bottom of the perforated base plate (6) arranged for oxygen

semipermeable film (7) are separated from one another, the base plate (6) having strip-shaped partition walls (8) arranged on the top (8), dividing the base plate (6) and meandering flow through the meandering perfusion chamber (4) ), the lower layer chamber (3) with inlets and outlets (9, 10) at different heights for the flow of gases, the meander perfusion chamber (4) with inlets and outlets for culture media (11, 12) and the overlay chamber (5) is provided with feed and discharge (13, 14) for the overlay atmosphere and with an outlet connection (15) with screen fabric (16) arranged in the interior of the overlay chamber (5) in front of the outlet and on the discharge (12) of the meander perfusion chamber (4) there is an overflow (17) for the discharge of the used cell broth, characterized in that the bottom surface (18) of the base plate (6) of the

Meander perfusion chamber (4), the width and the height of the channels (19) formed by the partitions (8) make contact between the

Forcing seed cells, depending on the amount of cells to be grown and depending on the amount of seed cells of the starting amount, have a freely selectable size.

2. Meander perfusion bioreactor according to claim 1-, characterized in that the meander perfusion chamber (4) from the base plate (6) with strip-shaped arranged on the top (8), the base plate (6) dividing and a meandering flow through the meandering perfusion chamber (4) with 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 meandering perfusion chamber (4) are selected such that in the flow thread, in those formed by the partition walls (8)

Channel, an averaged laminar upstream with a Froude number not greater than 0.005 and a bottom flow with a Froude number close to 0 is formed, which is both a homogeneous with increasing cell volume

Concentration of nutrients, as well as no flow-causing flow in the overflowing medium of the sedimented cells ensures.

3. Meander perfusion bioreactor 1, characterized in that the

Bottom surface (18) of the bottom plate (6) of the meander perfusion chamber (4) between 20 to 1500 cm ² and the height of the channels (19) arranged on the bottom plate (6) of the meander perfusion chamber (4) between 1 , 8 to 6 cm and the distance (A) between the partitions (8) of the channel (19)

freely selectable between each other and from the side and end walls of the meander perfusion chamber (4) between 2 and 25 mm.

4. meander perfusion bioreactor according to claim 1- 2, characterized

characterized in that the bottom surface (18) of the bottom plate (6) of the meander perfusion chamber (4) is between 30 to 500 cm ² and the height of the channels (19) formed by the partitions (8) is between 1.8 cm and 2 , 5 cm freely selectable.

5. meander perfusion bioreactor according to claim 1-3, characterized

characterized in that the distance (A) of the partitions (8) of the channels (19) from one another and from the side and end walls of the meander perfusion chamber (4) can be freely selected between 4 and 6 mm.

6. meander perfusion bioreactor according to claim 1, characterized in that over the overflow (17) for the cell broth (with shut-off devices, not shown), several meander bioreactors are connected to each other in a module.

7. meander perfusion bioreactor according to claim 1-5, characterized

characterized in that a height-adjustable overflow (17) for the used cell broth is arranged on the feed (11) of the meander perfusion chamber (4).