WO1997045534A1 - Methode de culture cellulaire - Google Patents

Methode de culture cellulaire Download PDF

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Publication number
WO1997045534A1
WO1997045534A1 PCT/EP1997/002475 EP9702475W WO9745534A1 WO 1997045534 A1 WO1997045534 A1 WO 1997045534A1 EP 9702475 W EP9702475 W EP 9702475W WO 9745534 A1 WO9745534 A1 WO 9745534A1
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Prior art keywords
cell
cells
vesicles
vesicle
mammalian cell
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PCT/EP1997/002475
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German (de)
English (en)
Inventor
Manfred Kubbies
Peter Dörmer
Petra Meissner
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Boehringer Mannheim Gmbh
Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh
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Application filed by Boehringer Mannheim Gmbh, Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh filed Critical Boehringer Mannheim Gmbh
Priority to CA002256567A priority Critical patent/CA2256567A1/fr
Priority to AU30269/97A priority patent/AU3026997A/en
Priority to JP09541488A priority patent/JP2000511048A/ja
Priority to EP97924948A priority patent/EP0910625A1/fr
Publication of WO1997045534A1 publication Critical patent/WO1997045534A1/fr
Priority to US09/197,470 priority patent/US6251672B1/en

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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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the invention relates to a method for the cultivation of sucker cells which require contact with cell surface proteins of other sucker cells for activation, differentiation and / or proliferation, and devices for the cultivation of these cells.
  • Human cells cultivated in vitro are required, for example, in adoptive immunotherapy with autologous or allogeneic cells. Also of great importance are efficient methods for culturing hematopoietic progenitor and stem cells, which can be transplanted to the patient, for example after radiation or chemotherapy.
  • Cytotoxic T-lymphocytes are responsible for eliminating pathologically altered body cells, such as virus-infected cells or tumor cells.
  • CTL Cytotoxic T-lymphocytes
  • the patient's lymphocytes are activated in vitro and then reimplanted. Such activation can take place, for example, by adding interleukin 2 (EL2) to "promiscuous killer cells" (Thiele, D et al, Immunology Today 10 (1989) 375-381). Such cells are then called lymphokine-activated killer cells (LAK Cells) (Rosenberg, Immunology Today 9 (1988) 58-62) When stimulated with EL2, however, LAK cells are also obtained which are directed against healthy cells of the body (Chen, B. et al.
  • EL2 interleukin 2
  • LAK Cells lymphokine-activated killer cells
  • lymphocytes to be activated are cultivated in the presence of autologous tumor cells (Mixed Lymphocyte Tumor Cultures, Fossati, G et al., International Journal of Cancer, 42 (1988) 239-245). Another method is described in WO94 / 23014 Thereafter, lymphocytes are activated in a co-cultivation with a mammalian cell line, while avoiding allogeneic stimulation, to form tumoricidal cells. Instead of the cell line described there, fragments or vesicles of this cell line can also be used
  • Suspended vital tumor cells or fragments thereof are usually used for the ex vivo activation of CTLs and have previously been appropriately inactivated by chemotherapy / radiation therapy.
  • this method has significant disadvantages.
  • the inactivation of the tumor cells is complex (radiation, handling of toxic substances) do not rule out the fact that vital tumor cells or DNA from tumor cells are carried into the transplant during the transplantation of the CTLs.
  • inactivation of cells can lead to receptor modulations.
  • the secretion of inhibitory molecules from e.g. inactivated, still living tumor cells is not excluded.
  • the geometric / mechanical problem of the optimal cell density from effector to activator cells (hit probability) is also complex and can only be determined empirically.
  • the immobilization of biological effectors on cell culture surfaces is used to activate cells and to cause them to proliferate.
  • anti-T cell antibodies are immobilized on cell culture vessels by means of non-covalent binding by preincubation.
  • Added T cells proliferate by binding / interaction of their CD3 receptors with immobilized (CD3) antibodies (Geppert, T.D., Lipsky, P.E., The Journal of Immunology 6, Vol. 138 (1987) 1660-1666).
  • Antigen-specific CTLs can be induced in a similar process by immobilizing MHC molecules alone (Waiden, P., et al., Nature, Vol. 315 (1985) 327-239) or embedded in synthetic, planar membranes ( Watts, TH, et al., Proc. Natl Acad. Sci. USA, Vol. 81 (1984) 7564-7568). Furthermore, T cell activation can be modulated by interactions with immobilized accessory molecules (Moy, V.T., Brian, A.A., J. Exp. Med. 175 (1992) 1-7).
  • cells can be immobilized on cell culture vessel surfaces by non-covalent binding. Binding of monocytes to FKS-coated culture vessels and pulsing with specific antigens leads to an improvement in antigen presentation with increased antibody production of the B cells after co-cultivation with peripheral blood lymphocytes (Jahn, S., et al., Allerg. Immunol. 33 (1987) 239-244).
  • chemotherapeutic agents can be included in such vesicles to increase the local dose to induce cell death (Brown, PM, Silvius, JR, Biochimica et Biophysica Acta 1023 (1990) 341-351)
  • the in vivo application of artificial antitumor vesicles (liposomes) for antigen immunotherapy also corresponds to the state of the art (Phillips, NC, et al, Liposomes in the Therapy of Infectious Diseases and Cancer, 1989, Alan R Liss, Ine (ed), S 15-24, Bergers, JJ, et al, Cancer Immunol Immunother. 34 (1992) 233-240, Papahadjopoulos, D , Gabizon, A, Annais of the New York Academy of Sciences, Vol 507, RL Juliane (ed), 1987, 64-74)
  • WO95 / 02040 describes a process for the cultivation of hamatopoietic progenitor stem cells using feeder layers from stromal cells. However, the production of such feeder layers is complex
  • the object of the invention is to provide an improved method for the cultivation of sucker cells which, during the cultivation, make contact with cell surface proteins of other cells
  • This object is achieved by a method for cultivating a first mammalian cell which, for cultivation, makes contact with cell surface proteins of a second mammalian cell, characterized in that said first mammalian cell in the presence of an immobilized vesicle of the second mammalian cell which is part of the natural surface of the second Contains mammalian cell is cultivated
  • a complete second mammalian cell can also be used.
  • a second mammalian cell which is modified with a first partner of a biological binding pair and is bound to a fixed carrier via the second partner of the biological binding pair is
  • the cells or membrane vesicles are suitably immobilized by incubation with a binding-capable carrier.
  • the binding takes place via a biological binding pair, such as streptavidin / avidin, sugar / lectin, a CD molecule (eg CD3 / anti-CD3- Antibodies), MHC molecules (class I or II) or digoxigenin / anti-digoxigenin antibodies.
  • a biological binding pair such as streptavidin / avidin, sugar / lectin, a CD molecule (eg CD3 / anti-CD3- Antibodies), MHC molecules (class I or II) or digoxigenin / anti-digoxigenin antibodies.
  • Mammalian cells which are suitable for cultivation by the method according to the invention are cells which, for their cultivation, take up a receptor and / or antigen contact with cell surface proteins of other mammalian cells. Bioequivalent cell surfaces are therefore required for cultivation.
  • Such surface proteins are, for example, MHC complexes, costimulatory signals or adhesion molecules (Mescher, MF, Immunological Reviews 146 (1995) 177; Herold, C, et al., MS-Medecine Sciences, Vol. 1 1, No 5 (1995) 669-680; June, CH, et al., Immunology Today 15 (1994) 321).
  • the cultivation of the mammalian cell can be an expansion, an enrichment of a specific cell type from a cell mixture, an activation, differentiation and / or proliferation of a mammalian cell.
  • the method can be used particularly advantageously for the multiplication of cytotoxic T cells and the differentiation and proliferation of hamopathic cells Progenitor and stem cells in granulocytes, monocytes, erythrocytes, megakaryocytes and lymphocytes
  • the cultured cells are genetically modified during the cultivation, for example by transfection with a vector or by transduction with a therapeutically relevant retrovirus (one hand, MPW, et al, Blood 81 (1993) 254, Nolta, JA, et al, J Clin Invest 90 (1992) 342)
  • a therapeutically relevant retrovirus one hand, MPW, et al, Blood 81 (1993) 254, Nolta, JA, et al, J Clin Invest 90 (1992) 342
  • Such ex vivo genetically modified cells can be used in the context of gene therapy
  • vesicles or fragments of cells to which a binding partner of a biological binding pair is bound are also suitable for co-cultivation.
  • Cells which are normally used for the co-cultivation of nipple cells are suitable as cells for producing such fragments or vesicles for example, stromal cells as feeder layers, thymus cells, antigen-presenting cells or other cells which carry the signals for T cell activation.
  • signals are essentially those which initiate cell contacts, such as adhesion (for example via CD11a, CD18, CD54), antigen-specific recognition (via the MHC complex via T cell receptor) or cost-reducing signals (e.g. via B7 / CD28). This can also include the presentation of growth factors (Toksoz, D, et al, Proc Natl Acad Sei USA 89 (1992) 7350)
  • Cell vesicles can be produced by the processes familiar to the person skilled in the art. Vesicles can be produced, for example, by hypotonic shock or by incubation with cytochalasin B. Further processes are described, for example, in WO94 / 23014. Vesicles are obtained by such production processes, the others Cells present parts of the native cell surface of the parent cell, but themselves Such vesicles consequently no longer have a cell nucleus. The cell nuclei are preferably separated from the vesicles by filtration (approx. 2-5 ⁇ m pore diameter). If vesicles which are modified with a binding partner of a biological binding pair are to be used, it is advisable modified the intact cells and then vesicles were formed from these cells
  • biotinylation of the whole cells Free amino groups of proteins located on the cell membrane are preferably covalently bound to biotin.
  • a suitable reagent for this is for example D-biotinoyl-y-aminocarboxylic acid N-hydroxysuccinimide ester (B ⁇ otm-7-NHS)
  • the cells or vesicles still have to be modified before immobilization (binding of a binding partner of the biological system to the surface) essentially depends on the binding pair used. For example, if the binding pair CD3 / anti-CD3 antibodies or lectin / If sugar is used, it is not necessary to modify the cells or vesicles before immobilization. In this case, it is sufficient if the carrier is coated, for example with anti-CD3 antibody or lectin. The cells or vesicles can then naturally on their surface Binding existing structures (CD3 or sugar) to the corresponding binding partner, which is immobilized on the carrier. Such a method also has the advantage that selectively defined vesicles can be bound.
  • vesicles can be produced from a PBMNC preparation and an anti- CD3 antibodies only bind T cells (CD3 +)
  • An analogous differentiation of Vesicles are also possible with other suitable surface markers.
  • a binding pair in which a binding partner occurs naturally on the surface of the cells or vesicles to be immobilized a binding pair in which none of the binding partners occur naturally on the surface of the cells or vesicles can also be used In this case, the cells or vesicles must be modified prior to immobilization.
  • the partner of the biological system to be bound is preferably covalently bound to the cell surface via an activated amino group.
  • binding pairs are, for example, biotin / streptavidin, digoxigenin / anti-digoxigenin antibodies. It is preferred here to use a pair of bonds whose affinity is very high
  • vesicles and carriers it is not necessary for vesicles and carriers to carry the other partner of the biological binding pair with the respective surface, but it is also possible for vesicles and carrier to have the same binding partner on the surface in this case
  • the other binding partner preferably in soluble form
  • the vesicles and carrier surface can be biotinylated, and the immobilization is carried out by adding soluble streptavidin.
  • bind CD3 protein to the surface of the carrier and bind the immobilization of vesicles which carry CD3 protein on the surface by adding a soluble antibody which is directed against CD3.
  • the vesicles can be modified with more than one binding partner.
  • the binding to the carrier takes place via a binding partner, while further substances can be bound to the surface of the cells or vesicles via the further binding partner.
  • Antigens are preferably used which can be used to increase the immunogenicity in tumor cells (activation antigens such as B7, CD40, MHC II or ICAM)
  • the tumor cell vesicles lack essential signal-inducing surface proteins.
  • Such surface proteins are normally present on the normal cells of tumor subjects, naturally or inducibly.
  • signal proteins are, for example, the costimulatory signal proteins of the B7 family or CD40, antigen-presenting receptors, such as MHC I and / or MHC II, and adhesion molecules, such as ICAM 1 or LFA-3, which are usually supplied by normal hamatopoietic cells
  • a mixture of vesicles from different cell populations is used, preferably a mixture which contains tumor cell vesicles and further vesicles which carry costimulatory, signal-inducing surface proteins.
  • Such further vesicles are preferably produced from hematopoietic cells Monocytes, macrophages, dendritic cells or B cells In general, such cells can be used for the production of the further vesicles which naturally or induce the missing signals or make them available.
  • Hybrid surfaces of mixed immobilized vesicles from, for example, tumor cells and normal cells are formed Cells In this way, CTLs can be effectively induced in the cocultivation, for example of autologous T cells from tumor patients
  • the cells which are selected for use in the production of vesicles with signal peptides can be obtained directly from body fluids or tissues such as blood and / or Bone marrow, obtained or purified. They can also be grown in vitro using suitable culture conditions. When producing mixed hybrid vesicle surfaces, the same proportions of the respective vesicles are preferably immobilized. Depending on the vesicle size and density of the vesicles to be immobilized, however, portions that differ from this can also be immobilized.
  • the mammalian cells to be cultivated can first be brought into contact with a first type of vesicles in a first step and can be contacted with a further type of vesicles in a further cultivation step. This is expediently carried out until sufficient stimulation is achieved.
  • the vesicles expediently have a size of approximately 10-30% of the first mammalian cell to be cultivated. If cocultivation is to take place with vesicles from different cells, these vesicles are preferably of a size such that simultaneous contact with at least two different vesicles is possible. With a cell size of approximately 10 ⁇ m, the vesicles are expediently less than 500 nm, preferably less than 100 nm in diameter.
  • such activating substances can also be bound to the surface of the cells or vesicles if they are only modified with a binding partner.
  • the cells or vesicles are immobilized in a first step via the binding partner on the carrier and in a second step the activating substances are bound to the still freely available binding partners on the surface of the cells or vesicles.
  • the cell or the vesicle is therefore first bound to the support via the biotin / streptavidin interaction and then added to the still free biotins on the surface of the cells or vesicles streptavidin-bound B7, or biotinylated B7 and soluble streptavidin are added .
  • Antibodies to MHC class I or MHC class II molecules can also be used as binding partners for cell surface receptors (if unmodified vesicles are used). Binding partner, preferably on supports coated with streptavidin or avidin. Such an incubation for immobilization can be carried out in a simple manner, at room temperature or at elevated temperature, for several hours. Then the immobilized part of the cells or vesicles is removed by washing.
  • tumor cells, vesicles of tumor cells or lymphocytes isolated via a Ficoll gradient are used as second sucker cells for cocultivation
  • vesicles can be used in, for example, stromal two-step cultures, since they can be combined with different cytokines or ECM proteins and the culture conditions can be varied as desired more controllable cultural approach
  • growth factors such as SCF, LL1, IL-3, IL-6, JX-11, EPO, G-CSF, GM-CSF, flt-2 / flk-3 can therefore be used in cocultivation etc admitted
  • such growth factors or factors which mediate a costimulating signal or an adhesion signal can be bound to streptavidin when the cell surface is biotinylated and thus bound to the cell surface. This is particularly advantageous when vesicles of Tumor cells are used that usually do not carry a costimulatory signal. In this case, it is particularly preferred, for example, to bind streptavidin-bound B7 to the cell surface. This can increase the immunoreactivity of tumor cells
  • the stromal cell lines L87 / 4 and L88 / 5 were cultivated in long-term medium as described by Thalmeier et al, Blood 83 (1994) 1799-1807 and WO 95/02040.
  • the stromal cell lines L87 / 4 and L88 / 5 were grown as single-layer cultures in plastic culture bottles (NUNC, Wiesbaden-Biebrich) using LTC medium with 10 " ° M hydroeortisone (HSS).
  • the cells were incubated at 37 ° C in a CO 2 incubator (air / 5% CO2), trypsinized weekly and with a starting concentration subcultivated from 3x10 ⁇ cells / 25 cm 2 culture bottle
  • the cells are detached with 0.25% trypsin (Gibco) (10 min, 37 ° C), the cell number is determined and washed in PBS (without Mg 2+ and Ca 2+ ) (10 min, 1200 rpm, RT)
  • the cell pellets (with L87 / 4 approx. 2-3x10 7 cells, with L88 / 5 approx. 5x10 7 cells) are resuspended in 1 ml PBS
  • biotin-7-NHS solution D - biotinoyl - ⁇ - aminocaproic acid - N - hydroxysuccinimide ester, Boeh ⁇ nger
  • D - biotinoyl - ⁇ - aminocaproic acid - N - hydroxysuccinimide ester, Boeh ⁇ nger Free amino groups on and on the cell membrane locate proteins, react with biotin-7-NHS to form a stable amide bond. Unreacted biotin-7-NHS is removed by washing twice with 50 ml PBS.
  • the biotinylated cells are resuspended in 1 ml prewarmed EBSS (Sigma). Then a 90% glycerol solution (37 ° C) is pipetted into 3 equal parts at 5-minute intervals to the cell suspension in order to obtain a 30% glycine ⁇ to get rin solution. All steps are carried out at 37 ° C. 5 minutes after the last glycerol addition, the cell suspension is cooled in an ice bath for 2 minutes. All further steps are carried out at 4 ° C. The cells are centrifuged off (10 min, 1200 rpm, 4 ° C.) and the supernatant is discarded.
  • the pelleted membrane vesicles are taken up in approximately 5 ml of medium and plated in an amount of 50 ⁇ l / well in streptavidin-coated 96-well plates Incubation time of 5-10 hours at 37 ° C in a CO 2 incubator (air / 5% CO2) the unbound vesicles can be washed off.
  • the cell culture plates coated with streptavidin can be very homogeneous with the biotinylated membrane vesicles ( ⁇ 2 ⁇ m), i.e. with very small and relatively even spaces. After 5-10 hours of incubation, the vesicles adhere to the plates via the biotin-steptavidin bond and remain as a uniform coating even after vigorous shaking or rinsing (FIG. 1).
  • the loading of the plates results in a homogeneous vesicle lawn, which was stable for at least 1 week.
  • the cells and vesicles were analyzed at different times during the preparation. 2B clearly shows that the cells - after labeling with FITC-conjugated streptavidin - are largely biotinylated (right lower cluster).
  • the control batch of non-biotinylated cells is shown in Fig. 2C.
  • the prepared vesicles (Fig. 2E) are all biotinylated.
  • the control batch of non-biotinylated vesicles is shown in Fig. 2F.
  • the individual vesicles were still clearly visible in the streptavidin plates and were still bound to the plates.
  • the vesicles could be kept in medium or buffer at 4 ° C. for at least 8 days without the ability to attach via the biotin-streptavidin binding having been reduced. After a short vortex, the vesicles could be used in a similar way to freshly prepared vesicles.
  • CD34 + cells The method used here is the immunomagnetic separation of CD34 + cells from mononuclear cells (MNZ) with the help of Dynabeads® (Deutsche Dynal GmbH, Hamburg).
  • MNZ mononuclear cells
  • Dynabeads® Deutsche Dynal GmbH, Hamburg
  • the precursor cells were labeled with paramagnetic beads (beads) covalently bound to CD34 antibodies, separated from the unlabeled cells in a magnetic field, and the beads were then detached from the cells with the aid of a specific antibody preparation (DETACHaBEAD®; Deutsche Dynal GmbH) .
  • Umbilical vein blood was diluted 1: 5 with RPMI (with 25 units / ml heparin and 10 ⁇ g / ml DNase) in order to ensure the most efficient yield of MNZ.
  • the cell suspension was layered on Ficoll-Paque® and centrifuged at RT for 30 min at 1600 rpm.
  • the cells of the integral phase were then washed twice (RPMI / heparin / DNase), the cells were taken up in RPMI / 10% FCS + DNase and the cell number was determined.
  • the M-450 CD34 beads were washed with RPMI / 10% FCS before use and mixed well with the cells in Eppendorf vessels.
  • the optimal ratio of beads to cells was 1: 1.
  • the cell-bead mixture was incubated at 4 ° C. for 45 min and slightly rotated in the process.
  • the marked cells were then separated by means of a Dynal MPC magnet by washing four times and taken up in 100 ⁇ l RPMI / 10% FCS.
  • the cell suspension was mixed well with one unit (10 ⁇ l) DETACHaBEAD® per 10 7 Dynabeads and incubated at RT for 60 min with gentle rotation.
  • the detached CD34 + cells were separated from the beads by means of the magnet by washing twice. The cell number was then determined and the progenitor cells fed to the various tests.
  • the stromal cell lines were seeded in a cell density of 104 / well in LTC medium with 10 " 6 M HSS in 96-well plates, incubated for 24 hours at 37 ° C. in an incubator, with 15 or 20 gray (cesium 137, gammacell radiation system ) irradiated and cultivated for a further 12-24 hours, then the CD34 + precursor cells (3.2.10.1.
  • methyl cellulose cultures were set up. For this purpose, semi-solid cultures with 10 4 to 2xl0 4 non-adherent cells per ml in 14% IMDM, 30% FCS, 1% BSA, 5% PHA-LCM, 10 " 4 M L-glutamine and ⁇ -thioglycerol, 0.98 each % Methyl cellulose, 3 units / ml EPO and 100 ng / ml KL These cultures were incubated in 1 ml volume in 35 mm tissue culture dishes at 37 ° C. (air / 5% CO2) and the colonies after 14 days using a Inverted microscope (Zeiss) counted.
  • Inverted microscope Zeiss
  • the number of GM-CFC when cocultivated on L87 / 4 or L88 / 5 was 465.5 or 116.3 clones. When co-cultivated on appropriate vesicles, 90 or 76.3 GM-CFC clones were grown. The number of BFU-E when cocultivated on L87 / 4 or L88 / 5 was 104.5 or 13.8 clones. When co-cultivated on corresponding vesicles, 27.5 and 52.5 BFU clones grew.

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Abstract

Une méthode permettant de cultiver une première cellule de mammifère, que l'on met en contact avec les protéines de surface d'une seconde cellule de mammifère, est caractérisée par le fait que l'on cultive la première cellule en présence d'une vésicule immobilisée de la seconde cellule, ladite vésicule contenant des parties de la surface naturelle de la seconde cellule, ce qui est avantageux pour la multiplication des cellules de mammifère.
PCT/EP1997/002475 1996-05-24 1997-05-15 Methode de culture cellulaire WO1997045534A1 (fr)

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CA002256567A CA2256567A1 (fr) 1996-05-24 1997-05-15 Methode de culture cellulaire
AU30269/97A AU3026997A (en) 1996-05-24 1997-05-15 Cell cultivation process
JP09541488A JP2000511048A (ja) 1996-05-24 1997-05-15 細胞培養方法
EP97924948A EP0910625A1 (fr) 1996-05-24 1997-05-15 Methode de culture cellulaire
US09/197,470 US6251672B1 (en) 1997-05-15 1998-11-23 Culturing mammalian cells in contact with cell surface proteins

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EP96108288 1996-05-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011007A2 (fr) * 1999-08-10 2001-02-15 Acorda Therapeutics, Inc. Dispositif bioartificiel destine a la proliferation de tissu, preparation et utilisations correspondantes
WO2002046766A2 (fr) 2000-12-06 2002-06-13 Ecole Polytechnique Federale De Lausanne Reactif bioanalytique, procede de production, plates-formes de detection et procede d'identification fonde sur l'utilisation du reactif bioanalytique

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023014A1 (fr) * 1993-03-31 1994-10-13 Boehringer Mannheim Gmbh Lymphocites t tumoricides
WO1995002040A2 (fr) * 1993-07-07 1995-01-19 GSF - Forschungszentrum für Umwelt und Gesundheit GmbH Nouvelles lignees cellulaires stromales provenant de la moelle osseuse humaine et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023014A1 (fr) * 1993-03-31 1994-10-13 Boehringer Mannheim Gmbh Lymphocites t tumoricides
WO1995002040A2 (fr) * 1993-07-07 1995-01-19 GSF - Forschungszentrum für Umwelt und Gesundheit GmbH Nouvelles lignees cellulaires stromales provenant de la moelle osseuse humaine et leur utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHIM. BIOPHYS. ACTA (1994), 1195(1), 149-56 *
CHEMICAL ABSTRACTS, vol. 121, no. 21, 21 November 1994, Columbus, Ohio, US; abstract no. 245262, CORLEY, P. ET AL: "Binding of biotinated-liposomes to streptavidin is influenced by liposome composition" XP002016919 *
LOUGHREY H C ET AL: "Characterisation of biotinylated liposomes for in vivo targeting applications.", FEBS (FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES) LETTERS 332 (1-2). 1993. 183-188, XP002016917 *
TOKSOZ, DENIZ ET AL: "Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor", PROC. NATL. ACAD. SCI. U. S. A. (1992), 89(16), 7350-4, 1992, XP002016918 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011007A2 (fr) * 1999-08-10 2001-02-15 Acorda Therapeutics, Inc. Dispositif bioartificiel destine a la proliferation de tissu, preparation et utilisations correspondantes
WO2001011007A3 (fr) * 1999-08-10 2001-09-20 Acorda Therapeutics Inc Dispositif bioartificiel destine a la proliferation de tissu, preparation et utilisations correspondantes
WO2002046766A2 (fr) 2000-12-06 2002-06-13 Ecole Polytechnique Federale De Lausanne Reactif bioanalytique, procede de production, plates-formes de detection et procede d'identification fonde sur l'utilisation du reactif bioanalytique
WO2002046766A3 (fr) * 2000-12-06 2002-11-07 Ecole Polytech Reactif bioanalytique, procede de production, plates-formes de detection et procede d'identification fonde sur l'utilisation du reactif bioanalytique

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AU3026997A (en) 1998-01-05
JP2000511048A (ja) 2000-08-29
EP0910625A1 (fr) 1999-04-28

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