EP0910625A1

EP0910625A1 - Cell cultivation process

Info

Publication number: EP0910625A1
Application number: EP97924948A
Authority: EP
Inventors: Manfred Kubbies; Peter Dörmer; Petra Meissner
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH; Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH; Roche Diagnostics GmbH
Priority date: 1996-05-24
Filing date: 1997-05-15
Publication date: 1999-04-28
Also published as: WO1997045534A1; JP2000511048A; AU3026997A; CA2256567A1

Abstract

A process for cultivating a first mammary cell which makes contact for cultivation with cell surface proteins of a second mammary cell, in which said mammary cell is cultivated in the presence of an immobilised vesicle of the second mammary cell forming part of the natural surface of the second mammary cell, is of advantage for the multiplication of mammary cells.

Description

Method of culturing cells

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.

The cultivation of human cells is of great importance for various therapeutic approaches. 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 (CTL) are responsible for eliminating pathologically altered body cells, such as virus-infected cells or tumor cells. In adoptive immunotherapy, 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. , Cell Imrnunol 118 (1989) 458-469). In a further method for adoptive immunotherapy, the 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. However, 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.

Furthermore, the 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. For example, 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).

Furthermore, 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).

The production of artificial lipid vesicles is state of the art. On the one hand, these liposomes can be loaded with proteins embedded in the lipid membrane for better cell targeting in vitro (Herrmann, SH, Mescher, MF, Proc. Natl. Acad. Sei. USA, Vol. 78, No 4 (1981) 2488-2492 ; Bloemen, PGM, et al., FEBS Letters 357 (1995) 140-144; Bergers, JJ, et al., Journal of Controlled Release 29 (1994) 317-327; Gregoriadis, G., Immunology Today, Vol. 11 , No. 3 (1990) 89-97). On the other hand, 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)

In the previously known methods, artificial liposomes / vesicles are used either in solution, by non-covalent binding on supports or in vivo. It is also known to use cells covalently labeled via a binding partner in a cell separation process in order to immobilize bound, specific cells from unwanted cells ¬ ter to be able to separate antibodies (EP-A 0 701 130)

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

Instead of a vesicle of a second mammalian cell, a complete second mammalian cell can also be used. In this case, a second mammalian cell is used 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.

The use of the binding partners streptavidin / biotin, avidin / biotin or derivatives of these compounds for the immobilization of cells or their vesicles has not previously been described. So far, the production of biotinylated, artificial liposomes, wherein biotin has been bound to lipids (Bayer, EA, Wilchek, M., Liposome technology, CRC Press, Inc., 1984, Vol. III, 127-135; Loughrey, HC, et al., FEBS 13102, Vol. 332, No. 1.2 (1993) 183-188). These were examined in their binding process on SA-labeled microtiter plates, the primary goal being the binding of these artificial biotin vesicles to SA-labeled cells in vivo (Corley, P., Loughrey, HC, Biochimica et Biophysica Acta 1195 (1994 ) 149-156).

It has been shown that the activation and differentiation of cells by other cellular binding partners requires a large number of interactions between cells, antigens and / or factors acting simultaneously (Herold, C, et al., MS-Medecine Sciences, Vol 11, No 5 (1995) 669-680; Clark, EA, Ledbetter, JA, Nature 367 (1994) 425-428; Mayordomo, JI, et al., Nature Medicine, Vol. 1, No. 12 (1995) 1297-1302 ). For this reason, the use of preferably biotinylated, immobilized cells or native membrane vesicles derived therefrom represents a decisive advantage since, surprisingly, all the necessary reaction partners are located on or in the membrane. Furthermore, such an artificial surface is a universal reaction system, since possibly missing partners e.g. in the case of mutated tumor cells, they can easily be additionally applied using biological binding pairs.

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 necessary complexity of the interactions of surface proteins for cell activation, cell differentiation and cell proliferation is particularly evident in the co-cultivation of bone marrow stromal cells with haematopoieti see stem cells for long-term cultivation the latter cells (Sutherland, HJ., Eaves, CJ, Culture of Hematopoietic Cells, Freshney, RJ, et al (eds.), Wiley Liss, N Y., 1994, pp. 139; Koller, M R., et al, Bioteehnology 1 1 (1993) 358) This also seems to apply to the differentiation of T cells from hematopoietic progenitor cells ex vivo, where cocultivation with fetal thymus cells / tissue is required (Dou, YM, et al, Thymus 23 (1994 ) 195)

According to the invention, 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

In a preferred embodiment, 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) Such ex vivo genetically modified cells can be used in the context of gene therapy

In addition to intact cells, 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. Such 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 μm 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

The binding of a binding partner of the biotin / streptavidin system or biotin / avidin system, preferably of biotin, is expediently carried out by 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 (Bιotm-7-NHS)

Whether and to what extent 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. For example, 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. In addition to 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. Such 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

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 For immobilization, the other binding partner, preferably in soluble form, must be added. For example, the vesicles and carrier surface can be biotinylated, and the immobilization is carried out by adding soluble streptavidin. It is also possible to 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.

In a further embodiment, the vesicles can be modified with more than one binding partner. In this case, 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. Preference is given to binding substances which are required for the activation, proliferation or differentiation of cells. 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)

However, there is a possibility that 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. Such 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

In a preferred embodiment of the invention, therefore, 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.

In a further embodiment, 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 μm, the vesicles are expediently less than 500 nm, preferably less than 100 nm in diameter.

In a further embodiment, such activating substances can also be bound to the surface of the cells or vesicles if they are only modified with a binding partner. In this case, 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. For biotin as binding partner, 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, for example, 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. In a preferred embodiment, tumor cells, vesicles of tumor cells or lymphocytes isolated via a Ficoll gradient are used as second sucker cells for cocultivation

An advantage of the method according to the invention is that 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

In a preferred embodiment, 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

In a further preferred embodiment, 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 following examples, publications and illustrations explain the invention, the scope of protection of which results from the patent claims. The methods described are to be understood as examples which describe the subject matter of the invention even after modifications

1 shows the homogeneous loading of streptavidin-coated cell culture plates

(96-well plates) with biotinylated vesicles from L88 / 5 bone marrow stromal cells (100x magnification)

2 shows the flow cytometric analysis of cells and isolated vesicles

AC: Intact L88 / 5 bone marrow stromal cells DF: Isolated vesicles from L88 / 5 bone marrow stromal cells A / D: Morphological analysis of the particles using forward and lateral scattered light (FSC vs SSC) B / F: Detection of cell- or vesicle-bound biotins of biotinylated L88 / 5 cells using FITC-labeled streptavidins (Fll). Dead cells or vesicles of dead cells are labeled with propidium iodide (F12) C / F: like B / F, only on non-biotinylated L88 / 5 cells

example 1

Cultivation of stromal 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

Long-term medium:

McCoy's 5a with 12.5% FCS, 12.5% HS, 1% sodium bicarbonate, 1% sodium pyruvate, 1% vitamins, 0.4% non-essential amino acids, 0.8% essential amino acids, 200 mM L-glutamine, 1% Penicillin / streptomycin, 10 " ⁴ M α-thioglycerol, 10 ^{~ 6} M hydroeortisone

Example 2

Obtaining the stromal cell suspension

After confluence is reached, 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

Example 3

Biotinylation of stromal cells

In order to mark the cell membranes with biotin, 10 μl each of biotin-7-NHS solution (D - biotinoyl - ε- aminocaproic acid - N - hydroxysuccinimide ester, Boehπnger) are added and shaken at RT for 10 min. 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.

Biotin 7 NHS solution:

5 mg Biotin-7-NHS in 250 μl DMSO (= 20 mg / ml).

Example 4

Vesicle production (modified according to Jett et al., J. Biol. Chem. 252 (1977) 2134-2143)

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 cells are burst by rapid addition of 1 ml of cold Tris buffer and brief vortexing (hypotonic shock). After 5 minutes of incubation in an ice bath, the lysed cells are centrifuged (10 min, 1000 rpm, 4 ° C). = Supernatant) is filtered with the aid of a 5 μm filter unit (Millex®-SV filter unit, Millipore). The pelleted cell debris are in turn resuspended in 1 ml of cold Tris buffer, vortexed and also filtered through a 5 μm filter unit achieves the separation of larger membrane particles and the cell nuclei and receives a homogeneous suspension consisting of relatively equal (<2 μm) membrane vesicles. The filtrates are combined and centrifuged (20 min; 6700xg; 4 ° C)

Glycerin solution:

90% w / v Earle's balanced sah solution (EBSS)

Tris buffer:

10 mM Tris-Cl, pH 7.4; 1mM MgCl ₂ , 1mM CaCl2

Example 5

Immobilization of membrane vesicles via a biotin-streptavidin bond

The pelleted membrane vesicles are taken up in approximately 5 ml of medium and plated in an amount of 50 μl / 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.

Plate technology:

The cell culture plates coated with streptavidin (sterile) can be very homogeneous with the biotinylated membrane vesicles (<2 μm), 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.

Analysis of the vesicles:

In order to check whether the prepared vesicles were actually labeled with biotin and how evenly this labeling was done, 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.

Even after an incubation period of at least one week at 37 ° C, the individual vesicles were still clearly visible in the streptavidin plates and were still bound to the plates. Likewise, 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.

Example 6

Co-cultivation of human CD34 + cells from umbilical cord vein blood or bone marrow on vesicle-coated 96-well plates in comparison to irradiated cell veins or plastic

Obtaining 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). For this purpose, 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 μg / 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 μl RPMI / 10% FCS.

In order to detach the beads from the cells, the cell suspension was mixed well with one unit (10 μl) 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.

DNase solution:

Dissolve 10 mg DNase in 1 ml PBS; filter sterile.

Two-step cultures:

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. and 3.2.10.2.) enriched from umbilical vein blood were immobilized on the various cell layers (l-2xl0 ³ CD34 + cells / hole) or on the Once a week, half of the medium used was removed and replaced by fresh medium.After the end of the culture period (here 2 weeks), all cells were harvested, the cell number was determined and used in methyl cellulose cultures. Culture of methyl cellulose:

To quantify the clonality of CD34 + lines that had been cultured together in different vesicle-coated 96-well plates or on different stromal layers, 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 α-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.

Clonal Growth Results:

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. Comparing the co-cultivation of CD34 cells on vesicles of the stromal cell line L87 / 4 to the bare plastic surface of cell culture vessels resulted in 21.3 GM-CFC and 640 BFU-E clones in vesicle co-cultivation and 16.3 GM-CFC and 217.5 BFU -E clones when cultivated on plastic surface.

Reference list

Bayer, E.A., Wilchek, M., Liposome Technology, CRC Press, Inc., 1984, Vol. III, 127-135

Bergers, J.J., et al., Cancer Immunol. Immunother. 34 (1992) 233-240

Bergers, J.J., et al., Journal of Controlled Release 29 (1994) 317-327

Bloemen, P.G.M., et al., FEBS Letters 357 (1995) 140-144

Brown, P.M., Silvius, J.R., Biochimica et Biophysica Acta 1023 (1990) 341-351

Chen, B., et al., Cell Immunol. 118 (1989) 458-469

Clark, E.A., Ledbetter, J A., Nature 367 (1994) 425-428

Corley, P., Loughrey, H.C., Biochimica et Biophysica Acta 1195 (1994) 149-156

Dou, Y.M., et al., Thymus 23 (1994) 195

Einehand, M.P.W., et al., Blood 81 (1993) 254

EP-A 0 701 130

Fossati, G. et al., International Journal of Cancer, 42 (1988) 239-245

Geppert, TD, Lipsky, PE, The Journal of Immunology 6, Vol. 138 (1987) 1660-1666 Gregoriadis, G., Immunology Today, Vol. 11, No. 3 (1990) 89-97

Herold, C, et al., MS-Medecine Sciences, Vol. 11, No. 5 (1995) 669-680

Herrmann, S.H., Mescher, M.F., Proc. Natl. Acad. Be. USA, Vol. 78, No. 4 (1981) 2488-

2492 Jahn, S., et al., Allerg. Immunol. 33 (1987) 239-244 Jett et al., J. Biol. Chem. 252 (1977) 2134-2143 June, CH, et al., Immunology Today 15 (1994) 321 Koller, MR, et al., Bioteehnology 11 (1993) 358 Loughrey, HC, et al., FEBS 13102, vol. 332, no. 1,2 (1993) 183-188 Mayordomo, J.I., et al., Nature Medicine, Vol. 1, No. 12 (1995) 1297-1302 Mescher, MF, Immunological Reviews 146 (1995) 177 Moy, VT, Brian, AA, J. Exp. Med. 175 (1992) 1-7 Nolta, JA, et al., J. Clin . Invest. 90 (1992) 342 Papahadjopoulos, D., Gabizon, A., Annais of the New York Aeademy of Sciences, vol. 507,

R.L. Juliane (ed.), 1987, 64-74 Phillips, N.C., et al., Liposomes in the Therapy of Infectious Diseases and Cancer, 1989, Alan

R. Liss, Inc. (ed.), Pp. 15-24 Rosenberg, Immunology Today 9 (1988) 58-62 Sutherland, HJ., Eaves, C.J., Culture of Hematopoietic Cells, Freshney, R.J., et al. (eds),

Wiley Liss, N.Y., 1994, pp. 139 Thalmeier et al., Blood 83 (1994) 1799-1807 Thiele, D., et al., Immunology Today 10 (1989) 375-381 Toksoz, D., et al., Proc. Natl. Acad. Be. USA 89 (1992) 7350 Waiden, P., et al., Nature, Vol. 315 (1985) 327-239 Watts, T.H., et al., Proc. Natl. Acad. Be. USA, Vol. 81 (1984) 7564-7568 WO94 / 23014 WO95 / 02040

Claims

claims

1 A method of cultivating a first mammalian cell which makes contact with cell surface proteins of a second mammalian cell for cultivation, characterized in that said first mammalian cell is cultivated in the presence of an immobilized vesicle of the second mammalian cell which contains parts of the natural surface of the second mammalian cell.

A method of cultivating a first mammalian cell which contacts cultivating cell surface proteins of a second mammalian cell, characterized in that said first mammalian cell is cultivated in the presence of a second mammalian cell or a vesicle thereof, which or which modifies with a first partner of a biological binding pair and is bound to a solid support via the second partner of the biological binding pair.

3. The method according to claim 1, characterized in that the vesicle is immobilized via a Binde¬ partner for one of its cell surface molecules

4. The method according to claim 3, characterized in that the Zelloberflaehenmoiekul is a CD molecule, MHC class I or MHC class II molecule.

5. The method according to claims 1 or 2, characterized in that the cell or the vesicle is biotinylated and bound to the solid support via streptavidin or avidin

6 Method according to claims 1-5, characterized in that on the immobilized cell or the vesicle before cultivation via the biological binding pair used for immobilizing the second cell or the vesicle or via another biological binding pair, a substance which activates, proliferates and / or causes differentiation of cells to which the second cell or the vesicle is bound

7 The method according to claim 5, characterized in that for the production of biotinylated vesicles first whole cells are biotinylated and then biotinylated vesicles are formed therefrom.

8. The method according to claims 1-7, characterized in that a tumor cell or vesicle of a tumor cell is used as the second cell.

9. The method according to claims 1-8, characterized in that a mixture of vesicles of different cell populations will be used as the second mammal cell.

10. The method according to claim 9, characterized in that the first mammalian cell is first cultivated in the presence of a first immobilized vesicle and then in the presence of a second immobilized vesicle.