DE102005063175A1 - Separation of cells and bio-particles and/or molecules, for animals and biotechnology and medical diagnosis, adds functionalized micro-particles for recognition and bonding to be separated by membranes - Google Patents

Abstract

Separating cells, bio-particles and/or molecules, comprises placing a mixture of them in a reaction vessel together with functionalized micro-particles, which recognize and bond with the required targets. One or more membranes separate the mixture with pore sizes which allow the cells and bio-particles and molecules through but block the micro-particles, with a continuous use of various tubes and membranes and pumps and valves.

Description

The The invention describes an apparatus and method for isolation of cells, bioparticles and / or molecules of liquids. With her you can under Use of suitable carriers and known immobilization method specific bioparticles be recognized and separated. Fields of application of the invention are Animals involved in biotechnology (including biological research) and medical diagnostics.

The Isolation of cells, bioparticles and / or molecules liquids is important in many technical fields, numerous procedures are known.

The Cell analysis and cell separation have been used for decades Fluorescence Activated Cell Sorting (FACS). It is the Method of choice for the analysis of specific cell populations by surface markers. The FACS application is prepared for the isolation of large numbers of cells. The medium containing the cells must be heavily diluted The separation time is relatively long for larger cell volumes and it prepares Problems with observing aseptic conditions. The procedure causes overall significant costs.

to Detection and isolation of bioparticles and cells have been around more than 10 years increasingly magnetic separation processes used. For this purpose, either the catcher molecules are loaded with iron or a Iron core-containing microparticles are coated with the capture molecule. The separation is carried out by a strong magnetic field. The immuno-magnetic separation, successfully commercialized u.a. by Dynal and Miltenyi Biotec, has proven to be a simple, relatively inexpensive method the cell separation established. Especially compared to Fow cytometry has become the magnetic separation for proven the isolation of relatively rare cell types, so e.g. For the isolation of fetal cells from maternal blood for prenatal diagnosis. Another application is the detection of tumor cells in the blood after surgical removal of the primary tumor to induce others Treatments.

For therapeutic purposes, syngeneic CD34 ⁺ peripheral blood stem cells from patients suffering from certain malignancies of the blood-forming system are routinely recovered for reimplantation. This is preferably done by the purification of leukocytes with specific antibodies coupled to magnetic beads. For cell fractionation during the blood / cell donation several systems are available that use the different size and specific gravity of the blood cells for a separation in the gravitational field (centrifugation).

One Disadvantage of all sorting methods is that they are not continuous carried out can be. That a blood or lymphocyte sample is taken, the cells are incubated with the immunomagnetic particles. After disconnecting and Washing, the cells are detached from the magnetic particle and then can for therapeutic Purposes are used.

One good overview of FACS and MACS is given in "Fow Cytometry and Sorting "(Ed et al., Wiley & Sons, Inc., New York, 1990).

Other methods of isolation and / or removal of cells are described in U.S. Patent Nos. 4,149,875 EP 12311956A2 . US 6900029 . US 6432630 . US 20020012953A1 . DE 10022635A1 . US 5246829 . US 5739033 . US 5763203 . EP 0016552A1 WO 00/38762 EP 0502213B1 and EP 0554460B1 ,

Of the Invention is based on the object, a simple system for separation of cells, bioparticles, and other molecules that develop for animals can be applied, as well as in biotechnology including biological research and in medical diagnostics.

The Invention is according to claims 1, 14 and 29-30 realized the subclaims are preferred variants.

The Invention describes a simple separation process that exists everywhere can be applied where for the agent of a mixture to be separated is a specific one Ligand functional to a surface can be immobilized. The actual separation is done by Sorting by particle size (filtration, Seven).

For the solution of this The task will be based on standard methods of coupling specific ligands a solid carrier used.

As solid carriers known biopolymers such as membranes or particles of organic or synthetic polymers are used. The surface may be biocompatible and consist of collagen, purified proteins, purified peptides, polysaccharides such as chitosan, alginate, dextran, cellulose, glycosaminoglycans or synthetic polymers such as polystyrene, polyesters, polyethers, polyanhydrides, polyalkylcyanoacrylates, polyacrylamides, polyorthoesters, polyvinyl acetates, block copolymers, Polypropyls, polytetrafluoroethylene (PTFE) or polyurethane. In addition, the polymers may contain lactic acid polymers or copolymers (lactic acid and / or glycolic acid (PLGA).

The used surfaces can biodegradable or non-biodegradable.

The used specific ligands for the binding of the target molecules on the surface of cells Naturally or synthetic nature, e.g. Antibodies, antibody fragments, peptides, polypeptides, Glycopeptides, soluble Receptors, steroids, hormones, mitogens, antigens, superantigens, Growth factors, cytokines, leptins, viral proteins, adhesion molecules or Chemokines.

For the specific Use at least one antibody or antibody fragment is used, wherein the specific ligands are covalently bound to them or via spacers or linkers are fixed to them.

The vehicle can have any geometry. Membranes as capillary or Particles offer the advantage of a large surface. For the procedure to be described are microparticles with a diameter of> 10 microns <800 microns, preferably 50-500 microns used. For others Tasks can but also larger or smaller particles are used.

The with specific ligands (e.g., antibodies to CD34 or CD1d, or against HIV gp120) activated microparticles are extracted with blood, that with usual Anticoagulants is treated, brought in contact ex-vivo. The happens in a special reaction room. Should the reaction space be placed in an extra-corporeal circuit, secure hose connections, Valves, filters and pumps associated with the safety technology, that the system with no side effects for the patient after appropriate Anticoagulation can be operated.

The Target cells are bound by the functionalized microparticles. The separation of the now loaded microparticles is done by hydrostatic Pressure using a membrane (sieves), preferably in the form a tube, which allows all blood components to pass unhindered (pore size> 10 μm <800 μm) but the Retains microparticles. there Filtration can be done by both vertical and tangential Pressure applied. The particles remaining in the lumen become returned to the reaction vessel or for analytical or preparative Derived purposes.

To Interruption of the blood supply, the reaction vessel for the replacement of the Cells are used by the microparticles. The so isolated, dispersed cells can be separated from the particles by the same procedure and now stand for diagnostic applications available.

A Another application is the targeted isolation of different cell types with the goal of co-cultivation, whenever the respective Cell types Surface molecules and / or Like biomolecules Need cytokines of other cell types, to your desired To fulfill function.

Around for the necessary cell-cell contact sufficient proximity of the desired Can reach target cells multiple specific ligands bound to a particle.

specific Cells for this application can et al T cells, B cells or stem cells.

The Process can be both batchwise and continuously carried out become. From the high variability in the particle design in terms of material, diameter and surface modifications There is a huge variety of possible applications in animals, in medical diagnostics, but also in biotechnology and biological research

Embodiment 1

Isolation of CD4-positive Cells from rat whole blood

ascites antibody (RIB 5/2) were using the Millipore Montage Antibody Purification Kit (LSK2 ABG 20) performed according to protocol.

The Purification was subsequently checked with an SDS denaturing gel (10%).

Coupling of the anti-CD4 antibody to the polymethylmethacrylate (PMA) particles

• 1. 1 ml PMA (particle diameter = 40 μm +/- 10 μm, 10 mg / ml; COOH / PEG-COOH modified,) centrifuged 2 min 3,000 × g, supernatant discard and take up in 1 ml of 0.1 M MES buffer pH 6.3.
• 2. 2 mg EDC and 2.4 mg N-hydroxysuccinimide in 0.5 ml 0.1 M MES Dissolve buffer pH 6.3 and add to the PMA particle suspension. 1 hour at room temperature with stirring incubate (activation of the particles).
• 3. Separate PMA particles by centrifugation and wash twice with 0.1 M MES buffer pH 6.3.
• 4. Activated PMA particles in 1 ml of 0.1 M MES Puf take pH 6.3 with 100-150 μg antibody; Coupling reaction at room temperature for 16 hours (overnight).
• 5. Neutralization of free binding sites by adding 100 μl of 1 M ethanolamine with following Incubation for 1 hour.
• 6. Separate by centrifugation and wash the functionalized PMA particles with PBS 3 times.
• 7. Incorporation of anti-CD4 PMA particles in 1 ml PBS pH 7.4 and storage at 4 ° C. Verification of antibody coupling by means of Goat-anti-mouse antibody PE ( 1 ).

Specific cell isolation of CD4 positive cells from whole blood

• 1. 4 ml of anti-coagulated rat whole blood with 1 ml (1 mg particles) of functionalized anti-CD4 PMA particles Suspension mixed.
• 2. Incubate for 60 min at room temperature.
• 3. Separation of anti-CD4 PMA particles (with and without bound Cells) from the whole blood by filtration of the blood-particle mixture by means of a special chamber with a cell sieve (40 μm).
• 4. Washing the anti-CD4 PMA particles with 30 ml PBS (1% FCS)
• 5. Take the particles out of the sieve chamber in approx. 1 ml PBS, Vortexing (mixing) for 15-20 Seconds.
• 6. Taking a particle sample for microscopic analysis ( 2 ).
• 7. FACS analysis of the leukocyte fraction in rat whole blood before and after treatment with the anti-CD4 PMA particles ( 3 and 4 ).

Replacement and qualification of the Cells from the particles.

• 1. anti-CD4 PMA particles become the adherent cells sedimented by careful centrifugation.
• 2. Take up the sediment in PBS, 2mM EDTA, 3mM mercaptoethanol and 20 U Papain.
• 3. Incubation with mixing for 30 min.
• 4. Separation of cells and particles by filtration using a special chamber with a cell strainer (40 μm); Washing the particles with 30 ml PBS (1 FCS); Absorption of the particles from the cell strainer in approx. 1 ml of PBS; Vortexing (mixing) for 15-20 seconds and repeating the washing.
• 5. Concentrate the isolated lymphocyte fraction by centrifugation 350 × g and analyze by FACS ( 5 ).

Embodiment 2

Isolation of a protein (IgG) from a cell lysate

ascites antibody (RIB 5/2) were using the Millipore Montage Antibody Purification Kit (LSK2 ABG 20) performed according to protocol.

The Purification was subsequently checked with an SDS denaturing gel (10%).

Coupling of the (mouse IgG ₂ ) anti-CD4 antibody to the polymethylmethacrylate (PMA) particles

• 1. 1 ml PMA (particle diameter = 40 μm +/- 10 μm, 10 mg / ml; COOH / PEG-COOH modified,) centrifuged 2 min 3,000 × g, supernatant discard and take up in 1 ml of 0.1 M MES buffer pH 6.3.
• 2. 2 mg EDC and 2.4 mg N-hydroxysuccinimide in 0.5 ml 0.1 M MES Dissolve buffer pH 6.3 and add to the PMA particle suspension. 1 hour at room temperature with stirring incubate (activation of the particles).
• 3. Separate PMA particles by centrifugation and wash twice with 0.1 M MES buffer pH 6.3.
• 4. Take up activated PMA particles in 1 ml 0.1 M MES buffer pH 6.3 with 100-150 μg antibody; coupling reaction at room temperature for 16 hours (over Night).
• 5. Neutralization of free binding sites by adding 100 μl of 1 M ethanolamine with following Incubation for 1 hour.
• 6. Separate by centrifugation and wash the functionalized PMA particles with PBS 3 times.
• 7. Incorporation of mouse IgG PMA particles in 1 ml PBS pH 7.4 and storage at 4 ° C. Verification of antibody coupling by means of Goat-anti-mouse antibody PE ( 1 ).

specific Isolation of Goat anti-mouse IgG from a cell lysate

• 1. 1 ml of cell lysate (HepG2, 4 × 10 ⁶ ) is mixed with 1 mg Goat anti-mouse IgG.
• 2. The protein mixture is functionalized with 0.1 ml (100 μg of particles) Mouse IgG PMA particle suspension added.
• 3. Incubate for 60 min at room temperature.
• 4. Separate the mouse IgG PMA particles (with and without the bound Goat anti-mouse IgG) from the cell lysate by filtration through a special chamber with a sieve membrane (10 μm).
• 5. Wash 3 times the PMA particle with 5 ml PBS each.

Replacement and qualification of the Protein from the particles.

• 1. Recording of the functionalized PMA particles with the adhering proteins from Siebkam in 0.5 ml citrate buffer pH 2.2; Incubation for 30 min.
• 2. Separation of the particles by filtration by means of a special chamber with a sieve membrane (10 μm); Wash the particles with 2 ml citrate buffer.
• 3. Analyze detached proteins in citrate buffer ( 6 ).

Embodiment 3

The 7 - 11 show examples of the apparatus realization of the invention or of parts thereof. 7 represents a system that is typically usable for continuous use. Dashed arrows represent the tube system, arrowheads mark the direction of flow of the fluid: Starting from the organism (mammalia), body fluid, for example blood, is pumped or passed through a blood pump and a valve (surrounded by a circle) into a reaction vessel that has been functionalized Contains particles which interact in a mixture with constituents of the body fluid, if appropriate ( 8th ). The medium with cell mixture and functionalized particles is then passed into a particle separation system. According to one example, the medium with cell mixture and functionalized particles is passed through a sieve and through a hollow-fiber membrane and separated into a cell mixture without functionalized particles and into a cell mixture with functionalized particles ( 9 ). While the depleted cell mixture without functionalized particles derived via a valve is supplied to the organism, the cell mixture with functionalized particles and the target cells / bioparticles / molecules bound thereto is returned to the reaction vessel ( 7 ). 10 exemplifies the principle of separation within the hollow fiber membrane. The medium with cell mixture and functionalized particles is passed through a hollow tube membrane, wherein the membrane is provided with a pore size (dashed line), which allows only the passage of the cell mixture, now substantially no longer containing the target cells / bioparticles / molecules, while the functionalized particles remain with the separated target cells / bioparticles / molecules in the interior of the hollow tube system.

In 11 there is shown in detail a corresponding particle separation system which can be used for the use of two types of functionalized particles of different diameter.

Legend to the pictures:

1 : Mouse anti-rat CD4 antibody coupled to PMA particles Detected with Goat-anti-mouse antibodies labeled with the fluorescent dye PE

2 : Particles coupled with anti-CD4 antibody after 1 hr incubation with rat whole blood and washing Detected with calcein AM as live staining

3 : FACS Analysis of whole blood cells before cell isolation using activated PMA particles labeled with anti-CD4-FITC (FL1) quadrant at lower left shows CD4 positive cells

4 : FACS analysis of whole blood cells after cell isolation of CD4 positive cells using activated PMA particles labeled with anti-CD4-FITC (FL1) quadrant at lower left shows CD4 positive cells

5 : FACS analysis of particle-detached cells labeled with anti-CD4-FITC (FL1) quadrant at lower left shows CD4 positive cells

6 PAGE cell lysate without IgG, cell lysate with IgG, isolated goat IgG from citrate buffer

1

cell lysate before protein purification

2

eluted Proteins from the Sephadex Beads

3

eluted Proteins from the Sephadex Beads

4

cell lysate before protein purification

7 Use of the device for isolating cells / particles / molecules from liquids (example: continuous, blood)

8th reaction vessel

1

mixture of cells / particles / molecules

2

functionalized particle

3

functionalized Particles with the adsorbed target cells / particles / molecules

9 Principle structure of the particle separation system

1

medium with cell mixture and functionalized particles

2

cell mixture without functionalized particles

3

cell mixture with functionalized particles

4

Hollow fiber membrane

5

scree

10 Hollow tube membrane of the particle separation system

11 Example of a separation system using two types of functionalized particles of different diameters

Claims (30)

Device for isolating cells, bioparticles and / or molecules from liquids with the aim of an application for Animals, in biotechnology (including biological research) and medical diagnostics, consisting of a. a reaction vessel made of biocompatible Material containing a mixture of cells, particles and / or molecules is, b. functionalized microparticles containing the desired Recognize and bind cells, bioparticles and / or molecule population, c. a particle separation system based on one or more Membranes with pores that do not contain the cells, bioparticles, and / or molecules but let the microparticles pass d. and at continuous Use of various hose systems, membranes, pumps and valves, around all acts of isolation, isolation, processing and Separation of cells, bioparticles and / or make molecules to be able to. Device according to claim 1, characterized in that that the specific cells, bioparticles and / or somatic molecules Cells, subcellular Structures, proteins and / or nucleic acids as well as possibly viruses, bacteria and / or protozoans and the fluids are Se and Exkrete, Blood, lymph, cerebrospinal fluid, or Contain suspensions of isolated cells from organ harvesting or Culture Media or fermentation solutions which contain cells, bioparticles and / or molecules. Device according to claims 1 and 2, characterized in that that the cell / bioparticle / molecules separation by the use of further functionalized microparticles with combined with the principle of adsorption of cells, bioparticles and / or molecules becomes. Device according to claim 3, characterized in that that the combined functionalized microparticles in their Size differ. Device according to claims 1-3, characterized that these functionalized microparticles have more than one specific Carry ligands and thus two and more different cells, Bioparticles and / or molecules be specifically adsorbed on the microparticle. Device according to claim 4, characterized in that that over the additional (s) Ligands can be a cell-stimulating function for living cells. Device according to claim 4, characterized in that that over the additional one Ligands are separated at least two different cell types, which through membrane / mediator interaction to stimulate at least lead to a cell type. Device according to claims 1-7, characterized that the functionalized microparticles in the liquid suspended and are freely movable. Device according to claims 1-8, characterized that the microparticles by mechanical action in suspension being held. Device according to claims 1-9, characterized in that that the liquid from the reaction chamber provided with the microparticles by filtration is freed from the microparticles. Device according to claims 1-10, characterized in that that for The filtration membranes are used, passing through pores with a diameter that did not adsorb all specifically Allow ingredients to pass, but not the microparticles used. Device according to claims 1-11, characterized in that that flat or hollow tube membranes with a pore diameter> 1 microns and <1,000 microns used for separation become. Device according to claims 1-12, characterized in that that the reaction vessel for the further Processing of isolated cells is used. Method for separating cells, bioparticles and / or molecules from liquids, characterized in that the cells to be separated by certain Surface antigens are characterized. A method according to claim 14, for separating cells, Bioparticles and / or molecules, characterized in that the surface antigen-recognizing principle a ligand natural or of synthetic origin. Method according to claim 15, characterized in that that the ligand is an antibody, an antibody fragment, a peptide, a polypeptide, a glycopeptide, a receptor, a steroid, a hormone, a mitogen, an antigen, a superantigen, a growth factor, a cytokine, a lectin, a viral protein, an adhesion molecule or a Chemokine is. Process according to claims 14-16, characterized in that the ligand is covalent or non-covalently bound to the microparticle. Method according to claims 14-17, characterized that as a solid carrier biocompatible polymers are used. Method according to claims 14-18, characterized in that the ligands are coupled to, preferably, sepharose. Method according to claims 14-19, characterized by a continuous or discontinuous separation of cells, bioparticles and / or molecules. Method according to claims 14-20, characterized that the reaction vessels with different functionalized microparticles as individual modules in any Number can be switched in series. Method according to claims 14-20, characterized that for the continuous separation of the functionalized microparticles introduced into the reaction vessel containing the cells, bioparticles and / or molecules Liquid is flowed through. Method according to claim 21, characterized that the liquid Blood is. Method according to claims 14-22, characterized that the cells, bioparticles and / or molecules of the microparticles through Application replaced special buffer become. Method according to claims 14-23, characterized that the cell release and, if necessary, the release of more Cells, bioparticles and / or molecules in the same reaction vessel takes place. Method according to claims 14-24, characterized that the cells, bioparticles and / or molecules through the same membrane filtration is separated from the microparticle suspension, as for the liquid separation is used. Process according to claims 14-25, characterized in that the separated cells, Bioparticles and / or molecules for analytical and / or further processing without the purpose of the application be used on humans. Process according to claims 14-26, characterized in that the separated cells, Bioparticles and / or molecules unprocessed for the purpose of research or treatment of diseases or modified into animals. Apparatus and method according to the preceding claims for ex vivo treatment of blood in an extra-corporeal circulation. Apparatus and method according to the preceding claims suitable for cleaning, accumulation or depletion of cells, bioparticles and / or molecules for diagnostics or further processing.