WO2002066598A1 - Systeme d'isolation automatique de cellules vivantes de tissus animaux - Google Patents

Systeme d'isolation automatique de cellules vivantes de tissus animaux Download PDF

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Publication number
WO2002066598A1
WO2002066598A1 PCT/DE2002/000590 DE0200590W WO02066598A1 WO 2002066598 A1 WO2002066598 A1 WO 2002066598A1 DE 0200590 W DE0200590 W DE 0200590W WO 02066598 A1 WO02066598 A1 WO 02066598A1
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WO
WIPO (PCT)
Prior art keywords
chamber
dissociation
tissue
cells
chambers
Prior art date
Application number
PCT/DE2002/000590
Other languages
German (de)
English (en)
Inventor
Frank W. Pfrieger
Original Assignee
Max-Delbrück-Centrum für Molekulare Medizin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10116155A external-priority patent/DE10116155A1/de
Application filed by Max-Delbrück-Centrum für Molekulare Medizin filed Critical Max-Delbrück-Centrum für Molekulare Medizin
Publication of WO2002066598A1 publication Critical patent/WO2002066598A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting

Definitions

  • the invention relates to a system which is fully automatic and standardized
  • Cells isolated from animal tissues play an important role in biomedical research and in the development of pharmaceuticals and therapeutic methods. They are used to create primary cell cultures that are used for functional tests, efficacy tests or other preclinical experiments (Animal Cell Culture Techniques, M. Clynes (ed.), 1998, Springer; In Nitro Models, D. Anderson, 1990, Drug Safety 5: 27-39), for the development of replacement tissue from stem cells and for the extraction of biological material such as D ⁇ S, R ⁇ S or proteins.
  • the purification of specific cells from tissues is becoming increasingly important with the rapid development of functional genome research. For example, certain genetic manipulations in transgenic animals can have an embryonic lethal effect, so that possible functions of the protein under investigation, which only occur in the postnatal animal, cannot be investigated in vivo.
  • the isolation and subsequent cultivation of cells from embryonic tissue allows studies of these functions in vitro.
  • Another task of functional genome research is the analysis of expression patterns of proteins in certain cells, for example a comparison of the protein patterns in different developmental stages or between sick and healthy tissue (e.g. tumor characterization).
  • the problem here is that each tissue contains a multitude of different cell types and that changes in the expression pattern in certain cells, which are only present in small numbers, cannot be detected due to the low amount of protein or messenger R ⁇ S.
  • This problem can be solved in that the biological material is not isolated from the entire tissue, but from a homogeneous population of the cell type relevant to the specific question obtained by prior dissociation and purification.
  • the object of the invention is therefore to enable a fully automated and standardized preparation of living cells with maximum yield.
  • the aim is to develop a modular system that can be adapted to various applications, from pure tissue dissociation to the purification of certain cell types that contain selectable features. Furthermore, it should be the loss-free processing of tissue samples that are only available in small quantities (e.g. biopsy material), the safe handling of risk material (e.g. infected tissue), the extraction of any large amount of cells through continuous operation and the possible combination with other applications, such as the subsequent transfection of the isolated cells using suitable devices.
  • tissue samples that are only available in small quantities (e.g. biopsy material), the safe handling of risk material (e.g. infected tissue), the extraction of any large amount of cells through continuous operation and the possible combination with other applications, such as the subsequent transfection of the isolated cells using suitable devices.
  • the system consists of the cavity composite shown in Figures 1 and 2. This consists of different sections, which can be combined depending on the application. First, in the dissociation section, the tissue association enzymatically and mechanically dissolved and a cell suspension obtained. In a subsequent cleaning step, undesired cell fragments and damaged cells in the subtraction section are removed from the suspension. Finally, desired cells can be isolated by an appropriate combination of subtraction and / or selection steps. The subtraction and selection of cells and cell fragments is carried out by recognition molecules (antibodies, lectins, peptides or the like) which bind specifically to molecules on cells or cell fragments. These recognition molecules can be coupled to magnetic particles, for example.
  • recognition molecules antibodies, lectins, peptides or the like
  • the cells loaded with magnetic particles are then immobilized in the subtraction or selection section by correspondingly strong magnetic fields.
  • the recognition molecules can be reversibly immobilized by binding to prepared surfaces.
  • the system is housed in a supply container, which contains the control electronics, storage containers as well as pumps, valves and hoses for the supply with the necessary solutions.
  • Those parts that come into contact with biological material or preparation solutions consist of inert, non-toxic and heat-resistant material.
  • the system should be equipped with sensors for temperature, pH, oxygen partial pressure and turbidity.
  • the system can also be designed in such a way that the parts that come into contact with cells are combined in one component and can therefore be replaced after a certain period of use.
  • the starting material for the preparation is tissue pieces or sections. These are introduced into the receiving chamber (1).
  • the chamber is closed by a closure (la), and the tissue is rinsed (3) by a liquid flow from the inlet (lb) via the outlet (lc) into a rotatably mounted drum (2) in the incubation chamber.
  • Figure 3 shows a possible version of the incubation chamber from the front.
  • the rotary movement of the cylinder during the incubation is driven by changing magnetic fields, which are generated by electromagnets (3 c) attached outside the incubation chamber (3) and act on permanent magnets (2a) located on the outside of the cylinder (2).
  • the cylinder contains inward notches (2b) which keep the tissue on it moving.
  • the cylinder is also provided with water-permeable pores (2c), which allow the preparation solutions to be exchanged, but which retain the tissue.
  • the cylinder has elevations (2d) on the outside which improve the mixing of the enzyme solution.
  • the preparation solution is used in the Incubation chamber through the inlet (3 a) and the outlet (3b) replaced by enzyme solution.
  • the tissue is then incubated in the enzyme solution.
  • the cylinder (2) is rotated slowly so that the enzyme solution is constantly mixed and the tissue is moved.
  • the incubation chamber can consist of a tube, the ends of which are connected to one another. The liquid in the tube with the tissue is then kept in motion during the incubation by external tube pumps.
  • the oxygen partial pressure and the temperature should be kept constant using appropriate sensors and control loops.
  • the temperature and fumigation of the incubation solution can take place in the storage container or directly in the incubation chamber.
  • the enzyme treatment is stopped by exchanging the enzyme solution via the inlet (3 a) and the outlet (3b) for an enzyme inhibitor solution.
  • the tissue pieces are brought into the dissociation chamber (5) by a liquid flow from the inlet (lb) via the transition chamber (4) (Fig. 1).
  • the dissociation chamber is then completely filled with solution via the inlet (4a).
  • the tissue bond is dissolved mechanically by repeatedly passing the tissue pieces through dissociation elements (6) which are attached to at least one point in the dissociation chamber (Fig. 4 - 6).
  • These dissociation elements exert shear forces on the tissue, which can be successively increased with repeated passages and thus enable the gradual release of individual cells from the tissue structure.
  • the diameter of the dissociation chamber is reduced around the dissociation elements so that the tissue pieces pass the dissociation elements with increased pressure.
  • the dissociation chamber can be constructed in two versions, which allow the repeated passage of the tissue pieces through the dissociation elements. Both versions are shown in Figure 4.
  • the dissociation chamber consists of an annular, closed tube, which can at least partially consist of hose material.
  • the solution with the tissue pieces comes into the chamber via the inlet (5a) and is circulated by propellers (5b) or externally attached peristaltic pumps, so that the tissue pieces repeatedly pass through the dissociation elements (6).
  • the propeller movement is driven by a magnetic field, which is generated by an electromagnet (5c) attached to the outside.
  • the dissociation chamber consists of a cylinder, which is sealed at the ends by movable pistons or membranes (5e).
  • the solution with the tissue pieces comes into the chamber via the inlet (5f) and is moved back and forth in the cylinder by the synchronous movement of the two pistons or membranes, so that the tissue pieces repeatedly pass through the dissociation elements (6) attached in the middle.
  • the end of the lower chamber which is realized by a piston or a membrane, is provided on its inner surface with a propeller (5g) or a similar device which is set in motion by an electromagnet (5h) attached outside the chamber.
  • the propeller whirls up the pieces of tissue before each upward movement of the piston. This ensures that all pieces of tissue pass through the dissociation elements and do not remain on the piston surface.
  • the dissociation elements which are to exert variable shear forces on the tissue, can be constructed in different fillings.
  • the dissociation elements consist of a series of rotatably mounted knives or wires (6a). In the first round, the knives are all in a row and roughly cut the tissue (position 1; Fig. 5). For the next rounds, the spaces between the knives that have to pass through the tissue parts become smaller and smaller (position 2-3; Fig. 5), so that the shear force acting on the tissue is gradually increased. The knives are locked in the various positions magnetically via the correspondingly positioned electromagnets (6b).
  • the dissociation elements consist of grids or perforated diaphragms (6c) and in version 3 (Fig.
  • These dissociation elements are realized in different variants (Fig. 6), which differ in the grid spacing, hole or constriction diameter.
  • element variants with ever smaller constrictions are introduced into the dissociation chamber one after the other.
  • a suitable set of element variants is housed in a magazine, which is realized in version 1 cassette-shaped (6e, Fig. 7) or in version 2 cylindrical (6f; Fig. 8).
  • the linear or circular movement of the magazines leads to the exchange of the elemental variations in the dissociation chamber.
  • the different element variants can be accommodated in different dissociation chambers, which the tissue must then pass through one after the other.
  • the narrowing diameters of the element variants used in each preparation must be adapted to the type of tissue. It applies that the narrower the diameter of the elements, the tougher the tissue.
  • the dissociation chambers contain at least one closable drain (5d, 5i; Fig. 4), which is provided with a sieve. This sieve only allows individual cells to pass through (pore diameter depending on cell size, approx. 10 to 50 micrometers). The sequence is such that only cells released from the tissue and floating in the solution flow away.
  • the process (5d, 5i) is opened with a certain delay after the dissociation has been interrupted. During this time, undissociated pieces of tissue can sediment on the bottom of the dissociation chamber. To do this, the dissociation chamber must be aligned along the gravitational field.
  • the detached cells then pass through a transition chamber (7; Fig. 1) into the middle mixing chamber (8; Fig. 9), which consists of an annular, closed tube. The solution volume emerging from the dissociation chamber is replaced by fresh solution.
  • the cell suspension located in the middle mixing chamber (8) is mixed with magnetic particles via the inlet (8a; Fig. 1). These particles have molecules on their surface that bind specific recognition molecules to cell fragments and dead cells.
  • the mixing chamber is completely filled with solution and the cell suspension is kept in motion during the incubation phase by propellers (8b) or by external peristaltic pumps.
  • the suspension is then brought into the tubular subtraction chamber (10) by an overpressure applied to the inlet (8a) via the collecting chamber (9).
  • This chamber has a magnetizable surface on one side, which is enlarged for the efficient depletion of undesired cell material.
  • a correspondingly large magnetic field is generated by electromagnets (10b) attached to the outside.
  • the magnetic field can also be generated by permanent magnets whose distance from the subtraction chamber can be changed.
  • the diameter of the subtraction chamber is reduced in the direction perpendicular to the unfolded side, so that the cells are brought as close as possible to the magnetized side, and cells with particles are efficiently intercepted.
  • intact, non-adherent cells are placed in one of the cells by applying an overpressure in the inlet (10c) via the distribution chamber (11) lateral mixing chambers (12a, 12b; Fig. 9) pressed.
  • the cell material immobilized in the subtraction chamber is then washed out of the inlet (10c) via the outlet (10d) by a liquid flow after the magnetic field has been switched off.
  • the subtraction chamber is thus available again for subsequent depletion steps.
  • the cells are mixed with magnetic particles again. These can either bind unwanted cells, which are then withdrawn from the suspension via the subtraction chamber, or recognize desired cells, which are retained in the selection chamber (13; Fig. 11, 12).
  • the cell suspension is brought into the selection chamber (13) via the collecting chamber (9) and the inlet (13a).
  • This chamber is built similar to the mixing chambers.
  • the cell suspension mixed with magnetic particles is set in circulating motion by propellers (13b). It contains a magnetizable immobilization zone (14; Fig. 11, 12) at at least one point, which the circulating cells pass repeatedly.
  • the desired particle-bearing cells can be gradually captured and removed from the suspension.
  • the immobilization zone can be separated from the rest of the chamber by controllable flaps (14a) and contains an inlet (14b) and outlet (14c).
  • the zone contains a greatly enlarged surface, which can be magnetized by an electromagnet (14d) attached to the outside.
  • This surface can be implemented in two versions. In version 1 (Fig. 11) it consists of a strongly branched, magnetizable catch arm (14e), which is embedded in the selection chamber. In version 2 (Fig. 12) it consists of a strong unfolding of the corresponding section of the selection chamber wall (14f) and an electromagnet (14g) attached to the outside.
  • the diameter of the immobilization zone in this embodiment is greatly reduced in the direction perpendicular to the unfolded side, so that cells are brought as close as possible to the magnetized side. As soon as a sufficient number of cells have been immobilized, the
  • the magnetic field is switched off and the cells are flushed by a liquid flow from the inlet (14b) via the outlet (14c) into an outlet (15) which can hold standardized vessels, such as centrifuge tubes.
  • cells and cell fragments can be immobilized in the subtraction and selection chamber via special surfaces in the chambers, which allow reversible binding of recognition molecules.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
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  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système qui permet de dissocier de manière entièrement automatique et standardisée des tissus animaux et d'isoler ensuite des cellules vivantes. Les domaines d'application de la présente invention sont la recherche et le développement en génie biomédical.
PCT/DE2002/000590 2001-02-20 2002-02-20 Systeme d'isolation automatique de cellules vivantes de tissus animaux WO2002066598A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE10107942 2001-02-20
DE10107942.7 2001-02-20
DE10116155A DE10116155A1 (de) 2001-02-20 2001-03-31 System zur automatischen Isolierung von lebenden Zellen aus tierischen Geweben
DE10116155.7 2001-03-31

Publications (1)

Publication Number Publication Date
WO2002066598A1 true WO2002066598A1 (fr) 2002-08-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2998744A1 (fr) * 2014-09-17 2016-03-23 Siemens Healthcare Diagnostics Products GmbH Dispositif d'incubation pour un appareil d'analyse automatique
EP2970856A4 (fr) * 2013-03-14 2016-10-26 Avita Medical Ltd Systèmes et procédés pour le traitement de tissus et préparation de suspension cellulaire à partir de ceux-ci
US10631974B2 (en) 2001-02-07 2020-04-28 Avita Medical Ltd Cell suspension preparation technique and device

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5035708A (en) * 1985-06-06 1991-07-30 Thomas Jefferson University Endothelial cell procurement and deposition kit
EP0446450A1 (fr) * 1990-02-09 1991-09-18 Thomas Jefferson University Dispositif pour collecter et traiter des tissus gras pour la production de produit cellulaire endothélial
WO1994003645A1 (fr) * 1992-07-31 1994-02-17 Thomas Jefferson University Dispositif et procede de traitement du tissu graisseux pour la production d'un produit a cellules endotheliales
US5409833A (en) * 1993-07-01 1995-04-25 Baxter International Inc. Microvessel cell isolation apparatus
US5786207A (en) * 1997-05-28 1998-07-28 University Of Pittsburgh Tissue dissociating system and method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5035708A (en) * 1985-06-06 1991-07-30 Thomas Jefferson University Endothelial cell procurement and deposition kit
EP0446450A1 (fr) * 1990-02-09 1991-09-18 Thomas Jefferson University Dispositif pour collecter et traiter des tissus gras pour la production de produit cellulaire endothélial
WO1994003645A1 (fr) * 1992-07-31 1994-02-17 Thomas Jefferson University Dispositif et procede de traitement du tissu graisseux pour la production d'un produit a cellules endotheliales
US5409833A (en) * 1993-07-01 1995-04-25 Baxter International Inc. Microvessel cell isolation apparatus
US5786207A (en) * 1997-05-28 1998-07-28 University Of Pittsburgh Tissue dissociating system and method

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10631974B2 (en) 2001-02-07 2020-04-28 Avita Medical Ltd Cell suspension preparation technique and device
US10729536B2 (en) 2001-02-07 2020-08-04 Avita Medical Ltd Cell suspension preparation technique and device
EP2970856A4 (fr) * 2013-03-14 2016-10-26 Avita Medical Ltd Systèmes et procédés pour le traitement de tissus et préparation de suspension cellulaire à partir de ceux-ci
US10626358B2 (en) 2013-03-14 2020-04-21 Avita Medical Ltd Systems and methods for tissue processing and preparation of cell suspension therefrom
EP3674396A1 (fr) * 2013-03-14 2020-07-01 Avita Medical Ltd. Systèmes et procédés pour le traitement de tissus et préparation de suspension cellulaire à partir de ceux-ci
US11124752B2 (en) 2013-03-14 2021-09-21 Avita Medical Ltd Systems and methods for tissue processing and preparation of cell suspension therefrom
EP2998744A1 (fr) * 2014-09-17 2016-03-23 Siemens Healthcare Diagnostics Products GmbH Dispositif d'incubation pour un appareil d'analyse automatique
JP2016061788A (ja) * 2014-09-17 2016-04-25 シーメンス ヘルスケア ダイアグノスティクス プロダクツ ゲゼルシヤフト ミツト ベシユレンクテル ハフツング 自動分析装置用の培養デバイス（ｉｎｃｕｂａｔｉｏｎｄｅｖｉｃｅ）
US9804180B2 (en) 2014-09-17 2017-10-31 Siemens Healthcare Diagnostics Products Gmbh Incubation device and methods for automatic movement of a reaction vessel therein for an automatic analysis apparatus

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