WO2005014801A1 - Ameliorations apportees a la productivite de cultures cellulaires - Google Patents

Ameliorations apportees a la productivite de cultures cellulaires Download PDF

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WO2005014801A1
WO2005014801A1 PCT/GB2004/003399 GB2004003399W WO2005014801A1 WO 2005014801 A1 WO2005014801 A1 WO 2005014801A1 GB 2004003399 W GB2004003399 W GB 2004003399W WO 2005014801 A1 WO2005014801 A1 WO 2005014801A1
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cells
dead
cell culture
cell
dying
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PCT/GB2004/003399
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Andrew Devitt
Christopher Darrell Gregory
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University Court Of The University Of Edinburgh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

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  • the present invention relates to methods and apparatus suitable for improving productivity of cell cultures.
  • the invention also relates to novel reagents for use in such methods and/or apparatus.
  • the present invention provides a method of improving productivity of a cell culture by removing dying, dead cells and/or parts thereof from the cell culture, comprising the steps of: a) providing a cell culture; b) subjecting the cell culture to conditions conducive to allow production of a desired product by the cell culture; c) contacting the cell culture with a reagent which is capable of preferentially binding to dying, dead cells and/or parts thereof from the cell culture; and d) removing at least a portion of said dying , dead cells and/or parts thereof from the cell culture which are bound to the reagent.
  • the method may effectively be a continuous one, with steps b) to d) being repeated as necessary and in any order, or continuously during the cell culturing and product production.
  • the cell culturing process and the steps of removing dead and/or dying cells may be carried out contemporaneously.
  • the method may also be carried out at the end of the cell culture process.
  • the method according to the present invention is intended for improving productivity of a cell culture. That is, the method is designed to improve an amount and/or quality of product produced by the cell culture in comparison to a cell culture not employing the present method.
  • Such products include biopharmaceuticals such as recombinant proteins and antibodies, which are routinely used today. It is often not possible to produce such complex products via synthetic means and the only way to produce them is by culturing cells which are capable of expressing such products. As the cost of producing such products can be extremely high, any degree of improvement can be seen as beneficial/advantageous.
  • the product could in fact be the cells themselves and in this manner removal of dead and/or dying cells may lead to higher titres and/or improved quality of viable cells being produced.
  • the cell culture comprises eukaryotic cells e.g. mammalian, such as human cells. It is understood that such cells can be fragile to excessive physical stress, such that the present invention is designed to impose reduced physical stresses to cells as may occur by previously known processes for removal of dead cells, which employ, for example, centrifugation steps. Generally the present invention does not include a centrifugation step. The skilled addressee will readily know what conditions are conducive to allow production of a desired product by said cell culture.
  • the cell culture is contacted with the reagent designed to remove dead and/or dying cells from said cell culture. Contacting under low shear forces may be achieved by for example, passing the cell culture over a substrate to which is adhered the reagent.
  • the substrate can be any suitable material which is generally applicable for use with the reagent to be adhered and includes for example glass, natural or synthetic polymers, metal and the like.
  • the substrate may be in the form of a flat planar surface, or could be for example the inner surface of a tube or the available surface of a foam material.
  • a cartridge, monolith or the like comprising a plurality of surfaces with adhered reagent may be used and the cell culture passed through the cartridge or monolith such that the culture can contact the reagent and dead and/or dying cells be removed from the culture.
  • the substrate, cartridge and/or monolith can be replaced as required.
  • the reagent may be coated and adhered on the surface of, for example, small beads, e.g.
  • Suitable methods of adhering the reagent to the substrate include natural (passive) binding, as many substrates such as plastics have a high binding capacity in their native state and molecules such as proteins adhere without needing to modify the surface of the substrate.
  • the substrate can comprise or be modified to include reactive groups on the substrate surface, e.g. carboxyl groups which can react with the free amines present in proteins and other amine-containing-biomolecules. Covalent coupling to the carboxyl groups can be achieved using for example carbodiimide reagents such as EDAC (l-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride) .
  • the substrate may also be coated with antibodies or other molecules that can specifically bind to the reagent to be adhered, or avidin that will bind to reagents comprising biotin attached thereto.
  • the reagent to bind dead and/or dying cells may be bound directly to the substrate or indirectly through intermediate molecule (s).
  • soluble CD14 fused to the Fc portion of, for example, human IgG (sCD14Fc) is used and this can be directly bound to the substrate, or the substrate can be coated with an anti- human-Fc, which will adhere to the sCD14Fc.
  • the substrate is also "blocked" as known in the art, with, for example, a non-specific protein such as bovine serum albumin, before passing cells over the substrate. It is also possible to render the beads magnetic such that the beads, once the dead and/or dying cells have been adhered thereto, can be separated from the body of the cell culture using a magnetic field or magnets.
  • the substrate can be reused and/or replenished. For example dead and/or dying cells may be washed off the substrate such that the substrate can be re-used or the substrate with adhered reagent could be replaced.
  • dead and/or dying cells and/or parts of dead and/or dying cells may be removed from the antibody using standard techniques known to individuals skilled in the art, for example washing the antibody-coated substrate with a solution of low pH (e.g. lOOmM glycine pH 2.8).
  • dead/dying cells are cells which are apoptotic (cells undergoing apoptosis) and/or necrotic (cytolysed or leaky cells) and include cell fragments such as apoptotic bodies or debris as a result of cell lysis and/or leakage.
  • necrosis can be defined by staining with annexin V and exclusion of PI. Other tests can be used to define apoptosis e.g. nuclear morphology, loss of mitochondrial membrane potential, activation of one or more caspases. Not all apoptotic cells will show all of these parameters at all times as they are cell dependent and dependent upon the stage of the apoptosis programme.
  • the amount of dead and/or dying cells in a culture is likely to vary depending on the type of culture, cells used, length of culture, type of product etc. However, any improvement in the amount of product produced as a result of removing dead and/or dying cells from the culture is encompassed by the invention. It may not be possible to remove all dead and/or dying cells or parts of dead and dying cells from culture, but desirably greater than 10%, for example 25%, 50%, 75%, 90% or 95% of whole dead and/or dying cells are removed. The removal may be accompanied by removal of debris.
  • the dead and/or dying cells are separated from the bulk or body of the cell culture.
  • the dead and/or dying cells may be removed from the cell culture or otherwise isolated from the viable cells in the cell culture, so as to not interfere with the viable cells.
  • the reagent which is capable of preferentially binding to dying and/or dead cells includes macrophages and acrophage-like cell lines e.g. THP-1, HL60, U937, Mono-mac 6; and J774; and cells expressing molecules capable of binding dead and dying cells e.g. Cos cells expressing recombinant membrane-associated CD14 (see Devitt et al (1998) .
  • Human CD14 mediates recognition and phagocytosis of apoptotic cells. Nature: 392 :pp505-509) . and proteins such as Annexin V (see Sigma catalogue and EP0755516) ; antiphosphatidylserine antibodies e.g 1H6 (Upstate UK Ltd.) and soluble CD14 (sCD14) , further described in detail hereinafter, or a combination thereof.
  • sCD14 and antibodies do not require Ca + for binding to apoptotic cells in contrast to Annexin V.
  • antibodies or other reagent which preferentially react to intracellular proteins or proteins expressed on the surface of dead and/or dying cells, but not to the same extent on the surface of living and/or viable cells, may also be utilised.
  • Such reagents would include antibodies to cytoskeletal components and other reagents such as the fungal toxin phalloidin. Reagents capable of binding to intracellular components may preferentially bind dead and dying cells as those cells lose their membrane integrity.
  • soluble CD14 is CD14 in any format that is not membrane associated and will include CD14 bound to surfaces (e.g. beads) and CD14 conjugated to reagents (e.g. fluorochromes) .
  • the present invention also relates to the use of reagents which are capable of preferentially binding dead, dying cells and/or fragments thereof for improving cell culture productivity.
  • the present invention also relates to new uses of sCD14 that have not hitherto been suggested.
  • sCD14 may be used in in vitro or in vivo experiments to identify apoptotic or dead cells. In vivo, it can be of importance to identify apoptotic cells. For example, tumours have areas of apoptosis within the tumour and certain tumours have many apoptotic cells. sCD14 could be labelled in some manner e.g. fluorescently (directly or indirectly conjugated) or isotopically e.g. Technetiu in order to allow imaging of the site of apoptosis. This may be of use in studying the efficacy of anti-tumour therapy (e.g.
  • sCD14 can be administered as a so-called "magic-bullet" when conjugated to a toxic/therapeutic moiety or prodrug that could be used to deliver the moiety or prodrug to site of dying or dead cells (e.g. tumours) . It may also be used to detect tumours or other sites of apoptosis in health screens. In vitro, it can be of importance to identify apoptotic cells. For example, researchers may wish to identify and/or quantify apoptotic cells within a culture. sCD14 could be labelled in some manner e.g. fluorescently (directly or indirectly conjugated) and by using appropriate detection technologies e.g.
  • reagents may be used as soluble factors or be expressed on cells. These reagents include: Integrins (e.g.
  • CD36 SR-A, MER, CD14, ABCA1, PSR, CR3 , CR4 , CD91/calreticulin, CD31, Fc ⁇ R, SHPS-1, SRB1, asialoglycoprotein receptor, thrombospondin-1, Gas-6, protein S, MFG-E8 , Del-1, ⁇ 2GPI, C-reactive protein and serum amyloid protein.
  • the present invention provides an apparatus for improved culturing of cells, the apparatus comprising a culture vessel for culturing cells, and a cell separation system for separating and enabling removal of dead and/or dying cells from a cell culture, the cell separation system comprising a solid substrate or substrates to which is/are adhered a reagent or reagents which is/are capable of preferentially binding to dead and/or dying cells of said cell culture, using, for example, a panning process (as defined below) .
  • the cell culture vessel and separation system conveniently in its own vessel are typically connected to one another, such that a cell culture medium comprising cells may be passed from the cell culture vessel to the separation system and thereafter back to the culture vessel.
  • the cell culture vessel and cell separation system may be separate and unconnected.
  • the cell separation system may simply be adapted to be added or incorporated within the cell culture vessel itself and removed, for example, for replenishing, as required.
  • the reagent or reagents capable of preferentially binding dead and/or dying cells is/are typically proteins, such as Annexin V, sCD14 or others mentioned hereinabove, macrophage, macrophage-like cell or other phagocytic cell (e.g. fibroblasts) , antibodies specifically reactive with a cell surface receptor or intracellular moieties which are present on/in dead and/or dying cells and/or combinations thereof.
  • panning refers to the process whereby a culture is brought into contact with the reagent (s) and substrate to facilitate removal of dead and/or dying cells and/or parts of dead and/or dying cells.
  • Figure 1 shows a schematic representation of a culture apparatus for use in a method according to one embodiment of the present invention
  • Figure 2 shows sCD14 binding to apoptotic human B cells.
  • the Burkitt lymphoma cell line Mutu I was induced to undergo apoptosis by UN irradiation (100 mJ/cm 2 ) , cultured for 16-18 h and labelled with sCD14-Fc (A) or sCD14-His (B) . Nominally, 50-100 ⁇ l of 20 ⁇ g/ml SCD14 was used to label 200,000 cells.
  • sCD14-Fc was visualised using goat anti-human ⁇ -chain-PE conjugate;
  • sCD14-His was visualised using mouse anti-CD14 mAb 63D3, goat anti- mouse-IgG-biotin, followed by streptavidin-PE. "Control' indicates non-irradiated cultures.
  • FIG. 3 shows sCD14 binding to apoptotic human T cells.
  • the human T cell line Jurkat was induced to undergo apoptosis by UN irradiation (A) or staurosporine treatment (B) . Cells were labelled and analysed after 16-18 h. Soluble ICAM-3 (sICAM-3) is used as a negative control protein; in (A), "Secondary” indicates labelling with goat anti-human ⁇ -chain-PE conjugate alone and "Control* indicates cells not induced to undergo apoptosis; Figure 4 shows sCD14 binding to apoptotic and necrotic human B cells.
  • A Cells were treated as in Figure 1 and labelled and analysed up to 18 h after irradiation. Histograms were created separately for apoptotic or viable cells according to light scatter properties.
  • BU12 is a mouse CD19 mAb and is visualised using biotinylated goat anti-mouse immunoglobulin, followed by streptavidin-PE (a positive control for staining) .
  • LPS-binding protein (LBP) was included with some samples at 1.2 ⁇ g/ml and is a protein that promotes binding of CD14 to LPS (one of its ligands) .
  • B Cells were induced into necrosis by incubating for 30 minutes at 56°C.
  • FIG. 5 shows BU65 hybridoma cells stained with AxV-biotin before and after removal of dead and/or dying cells with Annexin V-biotin and streptavidin-magnetic beads ;
  • Figure 6 shows improved productivity of Ab by hybridoma cell culture (9el0) depleted of dead and/or dying cells.
  • Hybridoma culture of 9el0 was panned on macrophages at a ratio of 20 non-viable cells per monocyte-derived macrophage (MDM) as per example 4.
  • MDM monocyte-derived macrophage
  • Productivity was assessed over time following depletion of non-viable cells;
  • Figure 7a shows anti-phosphatidylserine (anti-PS) Ab (1H6) staining of human B cells.
  • the human B cell line was stained using indirect immunofluorescence (standard methodology as used by people skilled in the art) and analysed using flow cytometry. Strong preferential staining of non-viable cells was noted. Using cytometry analysis techniques (as used by people skilled in the art) the FS/SS plots of events staining strongly with Ab 1H6 and weak/non-staining events were generated. Clearly the strongly stained events included dead and dying cells and particles smaller than whole cells (i.e.
  • Figure 7b shows agglutination of 1H6 coated beads by supernatants from cultures where cells had been removed by centrifugation- In this case sub-cellular debris caused beads to agglutinate
  • Figure 8 shows anti-phosphatidylserine (anti-PS) Ab (1H6) staining of hybridoma cells and debris of non- viable cells.
  • a hybridoma culture was stained using indirect immunofluorescence (standard methodology as used by people skilled in the art) and analysed using flow cytometry. Strong preferential staining of non-viable cells was noted.
  • FIG. 9 shows the effect of panning a culture of hybridoma cells on anti-PS Ab (1H6) or isotype control Ab. Ab was coated to polyether foam and cells sucked into the foam. After incubation unbound cells were cultured for 7 days and productivity assessed. Culture exposed to the non-viable and debris-binding Ab 1H6 showed improved productivity.
  • Figure 10 shows staining of dead and dying cells with reagents reactive with intracellular molecules.
  • a culture of human B lymphoma cells (induced to apoptosis with ionomycin) was stained with a monoclonal antibody to actin (AC15, Sigma Ltd) using indirect immunofluorescence (standard methodology as used by people skilled in the art) and analysed using flow cytometry (figure 10a) .
  • a hybridoma culture (9el0 cells) was stained with a fungal toxin (fluorescein-labelled) that is known to bind to actin (Phalloidin-FITC, Sigma Ltd) using direct immunofluorescence (standard methodology as used by people skilled in the art) and analysed using flow cytometry (figure 10b) .
  • FIG. 1 shows a schematic representation of a culture apparatus in accordance with an embodiment of the present invention.
  • the apparatus 2 has a culture vessel 4 for culturing cells 6, agitation of the culture medium 6 can be carried out by an impellor 8.
  • the culture medium 6 in which the cells are grown flows, together with the cells, along pipe 10 into a separation vessel 12.
  • a solid substrate 13 which has coated thereon a reagent or reagents (e.g. acrophages) designed to preferentially bind dead and/or dying cells
  • the cells in the culture solution come into contact with the reagent and dead and/or dying cells bind to the reagent whilst healthy viable cells substantially do not.
  • the culture medium 6 is then returned to the culture vessel 4 via pipe 14 and culturing continued.
  • the process of removing dead and/or dying cells from the culture medium 6 can be a continuous process during the culturing of the cells. That is, the culture medium can continuously be cycled through the separation vessel and returned to the culture vessel. In this manner dead and/or dying cells can continuously be removed from the cell culture.
  • the skilled man can easily appreciate what other features may be present in the apparatus, such as heating systems, O 2 /CO 2 control mechanisms, product extraction devices and the like. The skilled man will also appreciate that such a device need not be used in a continuous fashion as depicted.
  • Example 1 Preferential binding of SCD14 to apoptotic and necrotic cells;
  • the extracellular domains of CD14 (whole molecule lacking the GPI-anchor site and stop codon) were PCR amplified with restriction site (Hind III/Bam HI) .
  • This product was cloned (directionally) into the polylinker of a pCDM8-based vector named plgl (this is published in the reference below) .
  • a splice donor site was incorporated into the 3 ' end of the PCR product also so that the resulting pre-mRNA is spliced to yield a mature mRNA with the CD14 extracellular domain directly abutting the hinge region of the IgGl Fc.
  • the vertebrate splice donor concensus is a 5 * : C/AAGGTAAGT 3 ' ) .
  • This then produces a fused chimaeric protein with CD14 N- terminal to the Fc region.
  • This method and the plas id are published: D.L. Simmons. Cloning cell surface molecules by transient expression in mammalian cells. Chapter 5 in: Cellular Interactions in development: A practical approach. Edited by D.A. Hartley. A deposit of this construct has been made in accordance with the Budapest Treaty on the 1st August, 2003 and has the accession No. NCIMG 41188.
  • the protein is produced as follows: It was produced by transfecting the DNA into 293 cells using standard calcium phosphate-mediated transfection methods - see for example Sambrook et al: Molecular Cloning: A. Laboratory Manual (2000) . Cold Spring Harbor Laboratory Press) and after culture for 3-7 days the supernatent was harvested .
  • the produced recombinant protein was purified by binding to protein G in a column (Pharmacia Biotech) . After washing the column to remove impurities the desired protein was eluted at low pH using lOOmM glycine pH2.7 and neutralised by adding 50 ⁇ l 1M Tris at pH 9.0 per ml of eluate.
  • Protein was assessed for purity by western blot and ELISA and concentration of the protein was estimated by using a colorimetric technique (Bradford assay supplied by Bio-Rad) .
  • the present inventors have found that soluble forms of the CD14 molecule can be shown to bind preferentially to apoptotic and necrotic cells, as compared to viable cells. Results obtained are based on flow-cytometric assays of soluble recombinant human CD14 binding to human cells in suspension. Examples are shown in Figures 2-4. Two forms of soluble CD14 (sCD14) have been produced and analysed, one fused to the Fc portion of human IgG (sCD14-Fc) , the other tagged with multiple histidine residues (CD14-His) .
  • the protocol adopted for assessing binding of recombinant sCD14 to cells is immunofluorescence staining and flow cytometry (see Becton-Dickinson Manufacturers Protocols for details) .
  • cells are incubated with the recombinant protein, washed, and the bound recombinant protein is detected using an antibody (anti-Ig or anti-His) that is either directly conjugated to a fluorochrome or that is detected in turn by a tertiary reagent that is fluorochrome conjugated.
  • the labelled cells are run on a flow cytometer and histograms are generated that plot the relative number of cells (fluorescent events, on the y- axis) versus the fluorescence intensity (normally on a log scale, x-axis) .
  • histograms By comparing the histograms of cells subjected to different treatments and labelled with different recombinant proteins, it is possible to ascertain the capacity of sCD14 preparations to bind to viable, apoptotic and necrotic cells.
  • sICAM-3 as a control recombinant protein for comparison with sCD14.
  • FIGs 2-4 indicate that sCD14 binds with highest intensity to apoptotic or necrotic cells as compared with viable cells.
  • a human B cell line induced to undergo apoptosis by UN irradiation are compared with untreated (Control) cells.
  • Figure 2A shows preferential binding of sCD14-Fc, and 2B that of sCD14-His, to cells induced to undergo apoptosis. Differences in staining intensities are due to differences in concentrations of recombinant proteins and/or differences in visualisation protocols.
  • Figure 3 a human T cell line that has been subjected to similar treatments and analyses are shown.
  • apoptotic cells at time zero are present as a result of a low level of spontaneous, constitutive apoptosis that is a feature of these B cells.
  • Maximal labelling with sCD14 is observed at 12-15 hours post apoptosis-induction.
  • LBP which enhances binding of LPS to CD14
  • BU12 is a monoclonal antibody that binds to CD19 on the B cells and is included as a positive labelling control.
  • Soluble ICAM-3 and the secondary visualisation reagent are included as negative controls.
  • Example 2 Presence of apoptotic cells inhibits productivity in hybridoma cultures
  • apoptotic hybridoma cells were added to viable cultures and the effect on antibody (IgGl) production was assessed at 3 and 5 days.
  • the results show that antibody productivity is reduced by the presence of apoptotic cells in a dose dependent fashion (table 1) .
  • Hybridomas were from an exponentially growing culture containing 3.9% apoptotic cells. Control cells were from the same culture.
  • Annexin V was used to remove apoptotic cells from hybridoma cultures (figure 5) . Details how dead/dying cells were removed from cell culture using Annexin V coated magnetic beads are as follows: Hybridoma cells (BU65) were washed into binding buffer and stained with Annexin V-biotin as per manufacturer's instructions (Bender Medsystems) . Annexin V stained cells were removed by incubation with streptavidin-coated magnetic beads (Dynal Ltd) prior to exposure to a magnet.
  • apoptotic cells The level of apoptotic cells was assessed before and after the removal procedure using annexin V-FITC and flow cytometry as per manufacturer's instructions (Bender Medsystems) .
  • Table 2 the percentage of apoptotic cells was reduced from 5% to less than 0.2% or from 29% to 2% or less as follows: 4C/6 mouse hybridoma cells were exposed to UN irradiation (lOOmJ/cm ) 20 hours prior to study. A mixture of these cells (30%) and untreated cells (70%) or untreated cells alone, each at 5x10 /ml, were stained with Annexin V-biotin (Bender MedSystems Ltd) l ⁇ l per 10 cells in high calcium buffer for 15 min.
  • the cells were washed 3 times with high calcium buffer and resuspended in the same.
  • Various volumes of streptavidin-coated magnetic Dynabeads (6.7xl0 8 /ml in high calcium buffer) were added to 250 ⁇ l aliquots of cells in the wells of a 48 well tissue culture plate which was rocked on a GyroRocker STR-9 moving platform (Bibby Sterilin Ltd) sufficiently to keep the cells in suspension at room temperature for 30 min.
  • Reactants were transferred to 5ml of high calcium buffer in a 15ml polypropylene round bottom tube and AxV-binding cells were removed magnetically.
  • the proportion of apoptotic cells was determined by staining with DAPI. Table 2. Removal of apoptotic hybridomas by magnetic depletion of AxV binding cells
  • the present inventors wished to investigate alternative procedures that did not require a centrifugation step, since the present inventors have found that centrifugation itself has a detrimental effect on hybridoma cultures, the present inventors therefore sought a gentler method that would be more broadly applicable to a final product.
  • Example 4 Use of Macrophage monola ⁇ ers to remove apoptotic cells from hybridoma cultures
  • a human monocytoid cell line, THP-1 was used which, after differentiation to macrophage-like cells was used as a monolayer to capture apoptotic cells by "panning" .
  • THP-1 cells (10 6 /ml) were stimulated to differentiate by culture with phorbol myristate acetate (250nM) (PMA also known as TPA) and di-hydroxy-vitamin D3 (lOOnM) for 3 days. Both reagents are widely available (e.g. from Sigma) The differentiated cells form a confluent monolayer after this treatment and previously have been shown to be capable of the clearance of apoptotic cells.
  • PMA phorbol myristate acetate
  • lOOnM di-hydroxy-vitamin D3
  • Hybridoma cells were added to culture flasks of differentiated THP-1 cells. • Flasks were incubated at 37°C/5%C0 2 for various intervals with gentle rocking every 10 min. • Supernatants were tipped off and recovered hybridoma cells were counted, the percentage viability and percentage apoptosis were determined.
  • the percentage of apoptotic cells was reduced.
  • the efficiency of panning was increased by increasing the http: apoptotic cell ratio and the panning time (table 4) .
  • Example 5 Increased IgG Production by Hybridomas after Removal of Non-Viable/Apoptotic Cells by "Panning" on Human Macrophages
  • MNC Human peripheral blood mononuclear cells
  • Percoll Amersham-Pharmacia
  • Monocytes were enriched by adherence: MNC (10ml, 4x10 6 per ml) were incubated in Iscove's Modified Dulbecco's Culture Medium (IMDM) containing antibiotics and glutamine in 25cm tissue culture flasks at 37°C in a humidified atmosphere containing 5% C0 2 .
  • IMDM Iscove's Modified Dulbecco's Culture Medium
  • RPMI 1640 culture medium RPMI 1640 culture medium
  • MDM Monocyte-derived macrophages
  • Hybridoma Cells • 4C/6 (IgGl-producing hybridoma) or 9el0 (IgGl- producing hybridoma) hybridoma cells were cultured in RPMI-FBS. Exponentially growing stocks (viability > 93% as defined by Trypan Blue exclusion) were maintained by subculturing every 2-3 days. • Cells used for panning were not subcultured for 5-7 days in order to increase the proportion of dead and apoptotic cells. Immediately before panning, these "neglected" hybridomas were resuspended in prewarmed RPMI-FBS containing 10% (v/v) culture medium harvested from exponentially growing cells.
  • Hybridoma cells that were not attached to or ingested by MDMs were recovered by decanting culture medium then rinsing the cell monolayer with warm hybridoma CM (10ml) .
  • Viability of recultured cells and IgGl production was measured at intervals up to 11 days. IgGl was measured by single radial immunodiffusion using a sheep anti-mouse IgG antiserum and purified mouse IgGl as calibrator.
  • Hybridoma cells depleted of non-viable cells produced 31% more IgG than unpanned controls after 4 days culture; 43% more after 11 days (Table 6) And in a second experiment, 100% more after 14-23 days (figure 6) . Consistent with this, twice as many viable cells (3.5xl0 5 /ml) remained in cultures of panned cells compared with controls (1.7x10 5 /ml) after 11 days. Table 6 - Hybridoma IgG Production after Removal of
  • Non-viable/Apoptotic Cells by Panning on Human Macrophages IgGl concentration in culture supernatant ( ⁇ n )
  • Anti-phosphatid ⁇ lserine (anti-PS) antibody binds preferentially to dead and dying cells and parts of dead and dying cells ("debris').
  • the inventors have shown that an antibody directed to phosphatidylserine binds preferentially to dead and dying cells.
  • a population of a human B cell line was stained with anti-PS Ab.
  • the Ab binds preferentially to dead and dying cell zone cells (figure 7a) .
  • Staining of sub-cellular sized particles (debris) is evident (figure 7a) .
  • the inventors show that panning a culture of hybridoma cells on anti-PS antibody 1H6 improves productivity as follows:
  • Anti-Phosphatidylserine (Anti-PS) mAb Clone 1H6, mouse IgG2a from Upstate UK Ltd.
  • Anti-PS mAb was passively coated onto pre-wetted polyether form cores (0.5cm dia x 1.5cm) by overnight incubation of the core in a solution containing 50 ⁇ g/ml antibody in 0.05M carbonate/bicarbonate buffer, pH9.6 at fridge temperature .
  • Panning of hybridoma cells on foam coated with 1H6 was carried out by passing hybridoma cells through the antibody coated foam as follows:
  • Example 8 Binding of dead and dying cells with reagents reactive with intracellular components
  • the inventors have shown that an antibody directed to actin binds preferentially to dead and dying cells. Further they have shown that a non-antibody reagent reactive with actin similarly binds preferentially to dead and dying cells.
  • a population of a human B cell line was stained with anti-actin Ab (AC15, Sigma Ltd) . By presenting light scatter data from strongly staining cells it is clear that the Ab binds preferentially to dead and dying cell zone cells (figure 10a) .
  • reagents reactive with intracellular components is provided through the use of phalloidin, a fungal toxin well known to react to actin.

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  • Cell Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention se rapporte à des procédés et à des appareils permettant d'augmenter la productivité de cultures cellulaires. L'invention concerne également de nouveaux réactifs destinés à être utilisés dans de tels procédés et/ou appareils.
PCT/GB2004/003399 2003-08-04 2004-08-04 Ameliorations apportees a la productivite de cultures cellulaires WO2005014801A1 (fr)

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US60/492,369 2003-08-04

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007023181A1 (fr) * 2005-08-24 2007-03-01 Qiagen Gmbh Procede de separation de cellules vivantes
US7645739B2 (en) 2001-02-21 2010-01-12 Alavita Pharmaceuticals, Inc. Modified annexin compositions and methods of using same
EP2367930A1 (fr) * 2008-12-24 2011-09-28 Immunosolv Limited Technique de séparation de cellules

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0755516A1 (fr) * 1994-04-11 1997-01-29 Nexins Research B.V. Procede de detection et/ou de quantification et/ou de separation de cellules apoptotiques dans ou d'un echantillon
US6432653B1 (en) * 1989-06-29 2002-08-13 Aventis Pharmaceuticals Inc. Device and process for cell capture and recovery

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6432653B1 (en) * 1989-06-29 2002-08-13 Aventis Pharmaceuticals Inc. Device and process for cell capture and recovery
EP0755516A1 (fr) * 1994-04-11 1997-01-29 Nexins Research B.V. Procede de detection et/ou de quantification et/ou de separation de cellules apoptotiques dans ou d'un echantillon

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7645739B2 (en) 2001-02-21 2010-01-12 Alavita Pharmaceuticals, Inc. Modified annexin compositions and methods of using same
WO2007023181A1 (fr) * 2005-08-24 2007-03-01 Qiagen Gmbh Procede de separation de cellules vivantes
JP2009505646A (ja) * 2005-08-24 2009-02-12 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 生細胞の分離方法
US8222030B2 (en) 2005-08-24 2012-07-17 Qiagen Gmbh Method for the separation of living cells
US9506107B2 (en) 2005-08-24 2016-11-29 Qiagen Gmbh Method for extracting nucleic acid from blood
EP2367930A1 (fr) * 2008-12-24 2011-09-28 Immunosolv Limited Technique de séparation de cellules

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