DE202018000370U1 - Meander bioreactor for the isolation and propagation of cells from tissue sections of tumors, metastases and other tissues - Google Patents

Abstract

Meander bioreactor consisting of a rectangular bioreactor vessel (1) of preferably clear polymer material, which is sterile-closed with a lid (2), characterized in that the bioreactor vessel (1) is subdivided into three superposed chambers (3, 4, 5) and the lowermost chamber (3) as a lowerlay chamber, the chamber (4) arranged above the lowerlay chamber (3) as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) as Overlay chamber formed and the underlay chamber (3) and the meander perfusion chamber (4) by a perforated bottom plate (6) with a bottom of the perforated bottom plate (6) arranged semipermeable film (7) are separated from each other Oxygen preferably diffused through the film compared to other gases.

Description

The invention relates to a novel type of bioreactor, with which it is possible to grow in a single closed process step from small pieces of tissue certain cells both in the medium located in the bioreactor, as well as to activate the cells simultaneously, multiply and then separate from tissue residues , In particular, in this way tumor infiltrating autologous T lymphocytes (TIL) can be obtained from tumor tissue, metastatic tissue, its surrounding tissue and from lymph nodes affected by tumor cells.

The DE 695 34 132 T2 describes a cell culture device comprising a container having a cell culture compartment, the cell multiplication container being delimited by a lower, gas-permeable, preferably oxygen-permeable layer and an upper film which is selectively permeable to substances of a given size and impermeable to cells , The container is adapted to receive a culture medium between the upper foil and the lower gas permeable layer.

• - einem Basalmediumabteil , von dem sich mindestens ein Teil über der oberen Folie befindet, wobei ein Basalmedium auf der oberen Folie liegen kann;
• - nur einer einzigen Zugangsöffnung zu dem Zellkulturabteil;
• - einer Zugangsöffnung zu dem Basalmediumabteil; und
• - einem gasdurchlässigen Schichtträger , der unterhalb der gasdurchlässigen Schicht angeordnet ist und mit dieser teilweise in Kontakt steht, wodurch der größte Teil der gasdurchlässigen Schicht in einer im Wesentlichen horizontalen Lage gehalten wird, so dass sich Zellen einer Suspension entlang des horizontalen Abschnitts der gasdurchlässigen Schicht verteilen können und ein Gasaustausch mit dem Zellkulturabteil nicht wesentlich behindert wird.

The container consists of

• a basal medium compartment, at least a portion of which is above the top sheet, wherein a basal medium may rest on the top sheet;
• - only a single access opening to the cell culture compartment;
• an access opening to the basal medium compartment; and
• a gas-permeable support disposed below and partly in contact with the gas-permeable layer, whereby the major part of the gas-permeable layer is kept in a substantially horizontal position so that cells of a suspension are distributed along the horizontal portion of the gas-permeable layer and gas exchange with the cell culture compartment is not significantly hindered.

The supply of cells with medium is unregulated, the overflow of the cells is not directed.

• • Mittel zum Perfundieren eines flüssigen Zellkulturmediums durch die Zellkulturkammer;
• • Mittel zum In-Kontakt-Bringen des flüssigen Zellkulturmediums, das durch die Zellkulturkammer perfundiert wird, mit einer Sauerstoffquelle, so dass das flüssige Zellkulturmedium, das durch die Zellkulturkammer perfundiert wird, mit Sauerstoff angereichert wird, wobei diese Mittel zum In-Kontakt-Bringen eine für Gas permeable und für Flüssigkeit impermeable Membran enthalten;

und

• • Mittel zum kontinuierlichen, periodischen oder diskontinuierlichen Ernten nicht adhärenter Zellen aus der Zellkulturkammer durch die Strömung des Kulturmediums.

In the DE 693 32 374 T2 For example, a bioreactor for culturing human cells and / or tissue, including human stem cells or human hematopoietic cells, is introduced from a housing defining a cell culture chamber into the human cells and / or tissues, including human stem cells or human hematopoietic cells and wherein they may be cultured, wherein said cell culture chamber is configured to contain a plurality of inlet ports substantially equidistant from a plurality of outlet ports for passage of cell culture medium therethrough. The bioreactor continues to describe

• Means for perfusing a liquid cell culture medium through the cell culture chamber;
• Means for contacting the liquid cell culture medium perfused by the cell culture chamber with an oxygen source so that the liquid cell culture medium perfused through the cell culture chamber is oxygenated, which means for contacting a gas permeable and liquid impermeable membrane;

and

• • means for continuously, intermittently 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 incorporated into the housing such that the housing is divided into the cell culture chamber on one side of the membrane and into a gas chamber on the other side of the membrane. The bioreactor includes means for perfusing respiratory gases through said cell culture, comprising means for delivering the respiratory gases to the gas chamber whereby the respiratory gases flow through the membrane passing through the cell culture chamber and means for withdrawing spent respiratory gases from the gas chamber.

The invention according to the DE 69614535 T2 generally relates to a device for ex vivo storage and ex vivo culture of biological cells, and more particularly to a device of this type which stores and cultures the cells in a portable cassette while maintaining a sterile system that is closed to the outside environment.

• • ein Gehäuse,
• • eine Zellwachstumskammer innerhalb des Gehäuses, konfiguriert um eine Menge von biologischen Zellen und ein Wachstumsmedium zu tragen, wobei die Zellwachstumskammer eine Medium-Einlassöffnung und eine Medium-Auslassöffnung aufweist,
• • einen Mediumbehälter, der mit der Medium-Einlassöffnung der Zellwachstumskammer verbunden und konfiguriert ist, ein Wachstumsmedium zu enthalten,

und

• • einen Abfallbehälter, der mit der Medium-Auslassöffnung der Zellwachstumskammer verbunden und konfiguriert ist, von der Wachstumskammer abgelassenes Medium aufzunehmen, wobei die Zellwachstumskammer, der Mediumbehälter und der Abfallbehälter ein von der äußeren Umwelt abgeschlossenes steriles System bilden;

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

• A housing,
• A cell growth chamber within the housing configured to carry a quantity of biological cells and a growth medium, the cell growth chamber having a medium inlet port and a medium outlet port,
• A medium container connected to the medium inlet port of the cell growth chamber and configured to contain a growth medium,

and

• A waste container connected to the medium-outflow opening of the cell-growth chamber and configured to receive medium discharged from the growth chamber, the cell-growth chamber, the medium container and the waste container forming a sterile system sealed from the outside environment;

The cell growth chamber contains a preferably oxygen permeable and liquid impermeable membrane defining a portion of the cell growth chamber and a gas chamber located on the side of the membrane opposite the cell growth chamber. The portable cell growth cassette further includes a gas supply port carried 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 the CN1699558 (A ) relates to a large-scale killer cell culture technology for inducing hematopoietic stem cell factors by using a bioreactor to present umbilical cord blood hematopoietic stem cells, inducing hematopoietic stem cells in DC cells with cytokine, completing the function of the tumor antigen, to complete the selected activation of killer T lymphocytes with specific killing effect on tumors. The production of the Gelose microcarrier for the monoclonal antibody (CD3McAb), the activation of CIK cells and the continuous cultivation of CIK cells in the bioreactor are linked.

The DE 10 2011 106 914 B4 / EP25 43 719 A1 discloses a meander bioreactor for dynamic and stress-free expansion, differentiation and harvesting of the total fraction of hematopoietic cells, stem cells, progenitor cells, but also isolated, stimulated, activated fractions of these cells, except embryonic cells, where the bioreactor operates in perfusion mode. The bioreactor consists of a Bioreaktorgefäß whose bottom surface is provided with staggered partitions, which cause a meander-like guidance of the media with a laminar flow. The meander bioreactor can be connected in series via shut-off valves with other meander bioreactors in series. After being introduced into the bioreactor vessel, the cells form a coherent cell layer which almost covers the bottom surface of the meander bioreactor, the cells often having close contact with one another. The cell clumps resulting from the expansion are dissolved by shaking and harvested or are fed to a subsequent modular bioreactor with a larger bottom surface of the bioreactor vessel corresponding to the expansion rate of the previous bioreactor for further expansion.

There is no bioreactor described in the prior art, with the cells and especially TIL sterile technically safe and can be produced in therapeutically meaningful amounts due to their high cytotoxicity.

In none of the bioreactors for growing tissue or cells shown in the prior art, cells have hitherto been isolated, grown, activated, expanded and harvested simultaneously from tissues.

The object of the invention is to provide a meandering bioreactor which makes it possible initially to grow out of chopped tissue pieces (which originate from resulting surgical tissue parts or from biopsies) in a closed perfusion procedure, to multiply continuously, thereby to be activated and after Reaching a certain cell density can be harvested.

The object of the invention is achieved by a meandering bioreactor in which cells from the shredded tissue parts grow into the medium contained in a meander bioreactor, are activated and multiplied at the same time and separated from the tissue parts after reaching a specific density of adult and expanded cells.

The meander bioreactor according to the invention consists of a rectangular bioreactor vessel ( 1 ) of preferably clear polymer material, which is provided with a lid ( 2 ) is sterile closed. The bioreactor vessel ( 1 ) is in three superimposed chambers ( 3 . 4 . 5 ), the lowest chamber ( 3 ) as a sublay chamber overlying the underlay chamber ( 3 ) arranged chamber as a meander perfusion chamber ( 4 ) and those above the meander perfusion chamber ( 4 ) arranged chamber ( 5 ) are designed as an overlay chamber.

The underlay chamber (3) and the meander perfusion chamber ( 4 ) are by a perforated bottom plate ( 6 ) with a film tightly attached thereto and permeable to oxygen ( 7 ) separated from each other. At the sublay chamber ( 3 ) are Zu- and Discharges available ( 9 . 10 ), which allow a flow through with gases, in particular with mixtures of air and oxygen or with nitrogen and oxygen, in which the proportion of oxygen can be regulated. The oxygen diffuses preferentially over nitrogen through the gas-permeable film ( 7 ), which lie close to the perforated bottom plate ( 6 ) of the meander perfusion chamber ( 4 ) is attached. During the flow through the boundary-perfusion chamber ( 4 ), during the expansion of the cells, both from the overlay and from the sublayers (by diffusion) controlled amounts of oxygen in the culture medium.

The Meander Perfusion Chamber ( 4 ) with additions and exits for cultural media ( 11 . 12 ), as well as in the overlay chamber ( 5 ) Inlet and outlet (13, 14) for the overlay atmosphere and an outlet ( 15 ) arranged. At the outlet ( 15 ) is inside the overlay chamber ( 5 ) before the spout a mesh ( 16 ) arranged. At the exhaustion ( 12 ) of the meander perfusion chamber ( 4 ) is still an overflow ( 17 ) for the removal of the spent cell broth. The overflow is adjustable in height.

The Mander perfusion chamber ( 4 ) consists of the bottom plate ( 6 ) with on the top ( 8th ) arranged strip-shaped, the bottom plate ( 6 ) subdividing and a meandering flow through the meandering perfusion chamber ( 4 ) with gases and medium-enforcing partitions ( 8th ), wherein the distance (A) of the partitions ( 8th ) and from the side and end walls of the meander perfusion chamber ( 4 ) is selected so that in the current thread in which through the partitions ( 8th ) formed an average laminar upper flow with a Froude number <0.005. As the cell number increases (TIL or other cells), the supply of fresh medium is continuously increased, with the increased supply of fresh medium being automatically controlled by a corresponding algorithm. The bottom flow remains close to a Froude number 0 , whereby a Aufwirbelung of the cells is avoided. The described flow conditions prevent cell stress and ensure a homogenous supply of nutrients over the entire cell layer growing on the bottom of the channel.

The overlay chamber ( 5 ) has inflows and outflows ( 12 . 14 ) for the overflow with gases. The overlay chamber ( 5 ) is in each case with the same gas / gas mixture as the sublayers chamber ( 5 ) flows through.

In a culture run, cells usually grow out of the tissue pieces for 7 to 14 days; the adult cells multiply at the same time. Thus TIL can be produced in larger quantities from tumor tissue. In addition, tumor-infiltrating natural killer cells (TINK) and other cells can be extracted from tissues. When the glucose consumption in the bioreactor per day 100 reached up to 300 mg, the TILs are 16 ) provided outlet ( 15 ) of the bioreactor vessel ( 1 ) separated as filtrate from the tissue residues.

1. 1 Bioreaktorgefäß
2. 2 Deckel
3. 3 Unterlay- Kammer
4. 4 Mänder- Perfusions- Kammer
5. 5 Overlay- Kammer
6. 6 Bodenplatte
7. 7 Folie
8. 8 Trennwand
9. 9 Zuführung Unterlay- Atmosphäre
10. 10 Abführung Unterlay- Atmosphäre
11. 11 Zuführung Kulturmedium
12. 12 Abführung Kulturmedium
13. 13 Zuführung Overlay- Atmosphäre
14. 14 Abführung Overlay- Atmosphäre
15. 15 Auslaufstutzen
16. 16 Siebgewebe
17. 17 Überlauf für Zellbrühe

The meander bioreactor for the ex vivo recovery and propagation of cells from tissue parts are explained in more detail below, wherein the 1 a schematic sectional view of the meander bioreactor and the 2 show a schematic plan view of the meander bioreactor, wherein the reference numerals 1 to 17 Mark the design parts of the bioreactors listed in the following list:

1. 1 bioreactor vessel
2. 2 lids
3. 3 underlay chamber
4. 4 buttress perfusion chamber
5. 5 overlay chamber
6. 6 base plate
7. 7 foil
8. 8 partition
9. 9 feeder underlay atmosphere
10. 10 discharge underlay atmosphere
11. 11 feed culture medium
12. 12 discharge culture medium
13. 13 feeder overlay atmosphere
14. 14 exhaustion overlay atmosphere
15. 15 outlet spouts
16. 16 screen mesh
17. 17 overflow for cell broth

The starting material for the production of TIL or TINK and other cells are tissue components which are obtained, for example, during surgical removal of solid organ tumors, their metastases or their immediately surrounding tissue, or are taken for this purpose. A tissue amount of about 1 ml volume is usually sufficient as a starting material. The tissue samples are placed in a transport vessel with medium. The vessel is kept closed at room temperature and transported from the site of tissue removal into the clean room area for the preparation of the immune cell preparations. The surgical tissue pieces are crushed in a clean room under a LFB into pieces of 1 to 2 mm3 size and into a meandering Bioreactor transferred. The minced tissue pieces are evenly distributed in the bioreactor and cultivated in the perfusion mode. The bioreactor is connected ready for operation in a GMP Breeder and is continuously monitored, largely automatically controlled and documented by the Control Unit.

The meander bioreactor consists of a rectangular bioreactor vessel ( 1 ) of preferably clear polymer material, which is provided with a lid ( 2 ) is sterile closed. The bioreactor vessel ( 1 ) is in three superimposed chambers ( 3 . 4 . 5 ), wherein the lowest chamber as a sublayers chamber (3), the above the lower layer chamber ( 3 ) arranged chamber as a meander perfusion chamber ( 4 ) and those above the meander perfusion chamber ( 4 ) arranged chamber as an overlay chamber ( 5 ) are formed.

The underlay chamber (3) and the meander perfusion chamber ( 4 ) are by a perforated bottom plate ( 6 ) with a film tightly attached thereto and permeable to oxygen ( 7 ) separated from each other. At the sublay chamber ( 3 ) there are inflows and outflows ( 9 . 10 ), which allow a flow through with gases, in particular with mixtures of air and oxygen or with nitrogen and oxygen, in which the proportion of oxygen can be regulated. The oxygen diffuses preferentially over nitrogen through the gas-permeable film ( 7 ), which lie close to the perforated bottom plate ( 6 ) of the meander perfusion chamber ( 4 ) is attached. During the flow through the boundary-perfusion chamber ( 4 ), during the expansion of the cells from both the overlay and the underlay chamber (by diffusion) controlled amounts of oxygen in the culture medium.

The Meander Perfusion Chamber ( 4 ) with additions and exits for cultural media ( 11 . 12 ), as well as in the overlay chamber ( 5 ) Supply and discharge ( 13 . 14 ) for the overlay atmosphere and an outlet ( 15 ) available. At the outlet ( 15 ) is inside the overlay chamber ( 5 ) before the spout a mesh ( 16 ) arranged. At the exhaustion ( 12 ) of the meander perfusion chamber ( 4 ) is still an overflow ( 17 ) for the removal of the spent cell broth. The overflow is adjustable in height.

The Mander perfusion chamber ( 4 ) consists of the bottom plate ( 6 ) with on the top ( 8th ) arranged strip-shaped, the bottom plate ( 6 ) subdividing and a meandering flow through the meandering perfusion chamber ( 4 ) with gases and medium-enforcing partitions ( 8th ), wherein the distance (A) of the partitions ( 8th ) and from the side and end walls of the meander perfusion chamber ( 4 ) is selected so that in the current thread in which through the partitions ( 8th ) formed an average laminar upper flow with a Froude number <0.005. As the cell number increases (TIL or other cells), the supply of fresh medium is continuously increased, and this is automatically controlled by a corresponding algorithm. The bottom flow remains close to a Froude number 0 , whereby a Aufwirbelung of the cells is avoided. The described flow conditions prevent cell stress and ensure a homogenous 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 of gases. The overlay chamber ( 5 ) is in each case with the same gas / gas mixture flows through as the lower layer chamber ( 5 ).

In a culture run, cells usually grow out of the tissue pieces for 7 to 14 days; the adult cells multiply at the same time. Thus TIL can be produced in larger quantities from tumor tissue. But also TINK and other cells can be obtained from tissues. When the glucose consumption in the bioreactor reaches 100 to 300 mg per day, the TIL is 16 ) provided outlet ( 15 ) of the bioreactor vessel ( 1 ) separated as filtrate from the tissue residues.

From the transfer of tissue pieces into the bioreactor vessel ( 1 ) runs the entire process until filling of activated and expanded cells in a completely closed vascular system. Culturing 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 proliferation and activations succeed occasionally by supplements, which are fixed on the surfaces of the bioreactor clot. In the case of TIL and other immune cells, the proliferated and separated TIL are centrifuged, resuspended in freezing medium and aliquoted (approximately 50 million TIL per vial). The samples are stored in the gas phase over liquid nitrogen.

With the meander bioreactor according to the invention for the isolation and propagation of cells from tissue parts of tumors, metastases and other tissues, it is possible to grow certain cells into the medium contained in the bioreactor in a single closed process step from chopped tissue pieces, as well as the cells at the same time activate, multiply and then separate from tissue remnants. In particular, in this way tumor infiltrating autologous T lymphocytes (TIL) from tumor tissue, metastatic tissue, surrounding tissue and lymph nodes affected by tumor cells. The directed laminar flow over the cells with medium in the meander bioreactor leads to rapid growth of the cells. In the case of TIL, the cells which have specific cytotoxicity to the tumor cells from which or their environment they are obtained are preferably expanded. In a second method step, in a bioreactor which resembles the meander bioreactor of the first method step, but one can larger colonization area, therapeutically useful amounts of TIL are produced for clinical applications.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 69534132 T2
• DE 69332374 T2 [0005]
• DE 69614535 T2
• CN 1699558A [0010]
• DE 102011106914 B4 [0011]
• EP 2543719 A1 [0011]

Claims (4)

Meander bioreactor consisting of a rectangular bioreactor vessel (1) of preferably clear polymer material, which is sterile-closed with a lid (2), characterized in that the bioreactor vessel (1) is subdivided into three superposed chambers (3, 4, 5) and the lowermost chamber (3) as a lowerlay chamber, the chamber (4) arranged above the lowerlay chamber (3) as a meander perfusion chamber and the chamber (5) arranged above the meander perfusion chamber (4) as Overlay chamber formed and the underlay chamber (3) and the meander perfusion chamber (4) by a perforated bottom plate (6) with a bottom of the perforated bottom plate (6) arranged semipermeable film (7) are separated from each other Oxygen preferably diffused through the film compared to other gases. Meander bioreactor after Claim 1 , characterized in that the underlay chamber (3) with in different height attached inlets and outlets (9,10) for the flow of gases, the meander perfusion chamber (4) with inlets and outlets for culture media (11 , 12) and the overlay chamber (5) with inlets and outlets (13, 14) for overlay gases and with an outlet nozzle (15), on which inside the overlay chamber (5) in front of the outlet a screen mesh (16) is arranged, wherein at the feed (11) of the meander bioreactor chamber (4) an arranged overflow (17) for the spent cell broth is arranged in its height adjustable. Meander bioreactor after Claim 1 - 2 characterized in that the Mander perfusion chamber (4) from the bottom plate (6) arranged on the top (8) strip-shaped, the bottom plate (6) dividing and a meandering flow through the meander perfusion chamber (4) with medium enforcing partitions (8), wherein the distance (A) of the partitions (8) from one another and from the side and end walls of the meandering perfusion chamber (4) is selected so that in the current thread through which the partitions ( 8), an average laminar upper flow with a Froude number not greater than 0.005 and a bottom flow with a Froude number near 0 is set, which ensures both a homogeneous concentration of nutrients in the overflowing medium with increasing cell volume and causes no re-suspension of the sedimented cells. Meander bioreactor after Claim 1 - 3 characterized in that over the overflow (17) for the cell broth (with shut-off devices not shown) a plurality of meander bioreactors can be modularly connected to each other.

Images