CS276448B6 - Process for preparing permanent fused cells - Google Patents

Abstract

A method for producing permanent cell lines of animal and human origin by fusing normal cells of human and animal origin with biological components that sustain the culture in vitro by subjecting a normal human or animal cell to a karyoplasty, cytoplast, nuclear or cytoplasmic fraction rich in The mitochondria of lymphatic or epithelial tumor cells, which themselves are incapable of propagation and originate from transformed cells, are fused in the presence of polyethylene glycol or Sendai virus, and the cells obtained are grown in a nutrient-free medium.

Description

The invention relates to a process for the production of permanent cell lines of both animal and human origin. The cell lines thus obtained can be used to obtain cell products; For a long time, attempts have been made to permanently grow cells of animal and human origin independently of the original tissues, for both scientific and practical reasons. Until now, the results have not been satisfactory and only in some cases have it been possible to grow certain blood cells permanently.

It is known to use the so-called hybridoma technique for the production of monoclonal antibodies with defined antigen binding specificity. This procedure has been described in Kohler and Milstein's Continuous culture of fused cells secreting antibody of predefined specificity, Nature 256, 495-497 (1975) and can be used to obtain virtually immortal antibody-producing (AK) cells and to replicate these cells as desired. By fusing antibody-producing cells (B-lymphocyte) with a malignant cell (Myeloma), cell hybrids can be obtained that combine the properties of the two original cells, i.e., the ability to produce antibodies and the ability to grow permanently. The new word hybridoma was coined from the terms hybrid cell and myeloma.

To make it easier to understand the peculiarities of this technique, it is necessary to state the basics of the structure and synthesis of antibodies, for example immunoglobulin Ig. The immunoglobulin molecule consists of two identical L light chains and two identical H heavy chains, as shown in Figure 1. Each H and L chain is genetically and functionally divided into different regions. The dotted portions of the molecule are variable regions because there is a much higher degree of sequence heterogeneity in these regions than in other more stable regions. The antigen-antibody binding sites are located in the variable regions. The possibility of different amino acid exchanges creates great possibilities for the formation of three-dimensional structures that can be complementary to a large number of antigens. A mammal can usually create 10 ⁶ to 10? different antigen binding sites.

Antibodies are the product of B-cell synthesis. During the ontogenetic development of these cells from the basic stem cell, a number of variable gene regions combine with a relatively small number of stable regions, as in the case of L and H chains. Once genes are joined, a cell is formed it also passes the property to the daughter cells. In the absence of antigen, the B-cell remains quiescent. It produces and secretes only a small amount of immunoglobulin, but carries antibodies in the cell membrane that have the same antigen binding sites as the secreted antibody. Upon entry of the antigen into the body, a series of complex B-cell cell interactions occur.

B-cells whose membrane immunoglobulin specifically reacts with the antigen then divide to form daughter cell clones that differ from various other blood cells. In case the B-cell clone produces an antibiotic with the same structure and the same antigen binding site, the antidote is a monoclonal antibody. However, complex antigens, such as proteins, microorganisms or cells, contain a variety of antigenically active sites (determinants, epitopes) and therefore induce the formation of different B-cells, which divide and form different clones. This produces a large number of antibodies that differ in size, specificity and affinity. Thus, it is clear that the immune response will be polyclonal. It is known that the mouse produces up to 10 different antibodies against a single hapten, i.e. isolated determinants. Thus, it is clear that it is extremely difficult, if not impossible, to obtain a reproducible antiserum against a particular antigen. Efforts have been made for many years to obtain a procedure that would allow individual B-cell clones to be propagated in order to obtain a homogeneous, monoclonal antibody. For example, plasmacytone or myeloma, known as a malignancy in mice, rats and humans, has been used. Myeloma occurs when a B-cell malignants and multiplies unhindered, with the clone of the resulting cells producing large amounts of homogeneous antibody. Myeloma can be chemically induced in some strains of mice. All attempts to obtain a monoclonal antibody with known binding specificity against a particular antigen by hyperimmunization and induction of myeloma were unsuccessful. However, myeloma cell lines have emerged that can be grown in vitro and used to obtain hybridomas. The basic idea of Milstein and Kohler was to obtain a hybrid cell by fusing normal B-cells from immunized animals and a myeloma cell that grows permanently and can be grown.

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In the first experiments, myeloma cells were fused with mouse spleen lymphocytes, and the spleen was collected from mice immunized with sheep red blood cells. In this way, 10 viable hybrids were obtained, two of which produced antibodies specific against sheep red blood cells. The antibody-producing hybrids were so similar to myeloma cells that they grew continuously in culture and formed tumors upon implantation into mice. Of great practical importance was the fact that hybrid cells, like myeloma cells, could be stored in liquid nitrogen and maintained viable in this way for a long time.

Obtaining hybridomas

Mice are immunized in the same way as in the production of a standard antiserum, this is repeated every few weeks. Immediately before fusion, the mouse is sacrificed and the spleen is removed antiseptically. MM R

The spleen sheath is cut and the marrow is carefully pushed out. The marrow cells, i.e. about 10 spleen lymphocytes, are suspended in nutrient medium and mixed with myeloma cells in a ratio of 1: 1 to 10: 1. , i.e. a 30 to 50% solution of polyethylene glycol or suspended inactivated Sendai virus. After washing the medium, the cell mixture is transferred to sterile vessels at a concentration of 10 cells per ml and cultured in an incubator in the presence of carbon dioxide. 2 to 4 weeks after fusion, the growth of hybridoma clones is microscopically evident. From then on, it is also possible to monitor the presence of antibodies in the supernatant. For this purpose, the analysis must be carried out in such a way that antibodies in an amount of less than 1 [mu] g can be detected (RIA, ELISA, Immunofluorescence). Cells from positive partial cultures are then cloned, i.e. cultures with one cell type are established. Isolated clones that produce the desired antibodies are further grown and their ability to induce tumor can be demonstrated by injecting a suspension of cells into the abdomen of mice pre-treated with Pristan, usually Balb C inbred mice. 6 to 20 days after inoculation or from the fluid that is formed from the abdominal cavity to obtain antibodies, it is usually 50 to 150 mg of monoclonal antibody from one mouse.

Immediately after fusion, a very heterogeneous mixture of hybrid cells and cells that did not participate in the 8 3 fusion is obtained. A maximum of 10 viable hybridoma cells can be counted using 10 mouse spleen cells. Hybrid cells need some time before they proliferate, whereas cells that did not participate in the fusion grow immediately further, so the mixture must be selected to ensure the survival of a small number of hybridoma cells. The standard procedure for the formation of hybridomas is the so-called HAT-selection medium, known from Littlefield, JW: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710 (1964). In the name of the medium, it means Aminopterin, a substance that antagonizes folic acid and blocks the main pathways of DNA synthesis. Normal cells bypass the aminofterin block with thimidine cyanase TK and hypoxanthine guanine phosphoribosyltransferase ₍ HGPRT) if they have thymidine T and hypoxanthine dostupný available in the culture medium. uses a mutant of myeloma cells that do not contain TK or HGPRT, which survive in the HAT environment only if they fuse with a normal cell that brings the missing enzyme into the hybrid cell. viability in culture and therefore does not inhibit the growth of hybridoma cells.

The following problems occur when hybridoma cells form:

1. Selection using HAT environment

Selective suppression of myeloma cell growth is, as noted above, an essential condition for the generation of a hybridoma clone. However, selection using this medium is non-physiological even for normal cells, so it changes the ability to survive and the ability to reproduce. Especially when using human lymphocytes, it is very difficult to choose the concentration of the individual components of the HAT medium so that HGPRT-negative cells are killed, while HGPRT-positive cells can survive.

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The ratio between the number of lymphocyte cells and myeloma cells and the generation of hybrids, on the other hand, is such that using 10 mouse lymphocytes in a typical procedure, the generation of 500 hybridoma clones is a very good result. Because in the spleen and in the event that the mouse was immunized repeatedly produces antibodies only 10 ⁴ to IQ · ⁵ cells as described by Jerne NV and Nordin AA, Science 140, 405 (1963) is needed in hybridoma formation 10 ^ to 10 ⁴ clones in order to obtain a single clone with the desired antibody specificity. II of the human hybridoma, the ratio is even worse, and obtaining 4 to 10 hybrid clones in a single fusion can be considered a good result.

2. Chromosome loss

After fusion, the new hybrid cell must have a double number of chromosomes. As it turns out, hybrid cells tend to lose chromosomes. In each cell division, if the chromosome is too large, these chromosomes do not divide evenly into the two daughter cells. A daughter cell that has a smaller number of chromosomes has an advantage over other cells in selection and becomes the predominant cell in culture. However, the synthesis of immunoglobulin is not essential for the viability of the hybrid cell, i.e. it means that the excess chromosome that is involved in this luxurious synthesis can be removed. . Thus, the emergence of variants in a hybridoma clone that do not produce antibodies is a very common phenomenon and a number of measures, especially repeated selection, are needed to ensure the clone's ability to produce antibody. The tendency to lose chromosomes is particularly high in hybrid cells from unequal species. -

3. Hybrid immunoglobulins

Myeloma cells are malignant B-cells and themselves produce immunoglobulins with unknown antigen binding specificity. This ability is brought by the myeloma cell as well as the normal B-cell into the hybridoma. Because the different chains of Ig molecules are synthesized separately and only then assembled into finished antibodies, the hybridoma cell synthesizes two different L and H chains and about 10 possible combinations, of which the correct combination makes up only 1/16 of the total. Ig. For this reason, mouse myeloma cells have been very laboriously developed as mutants that do not themselves produce any H or L chain. However, no similar myeloma cell line is yet available for human lymphocyte fusion.

Other problems arise with other possible hybridoma formation processes.

I. Immortalization of B-lymphocytes by viruses

Human B cells from a normal donor can be transformed into a malignant infection with Epsteine-Barr virus (EBV). EBV-infected cells are lymphoblastoid and can be grown continuously in vitro and cloned. Compared to the hybridoma, lymphoblastoid EBV lines produce only 1/10 or less of the immunoglobulin with unsatisfactory production stability. It would be desirable to fix B-cells at an earlier stage of differentiation with EBV to obtain a more frequent occurrence of clones that produce small amounts of IgM. .

In an analogous manner, mouse B cells can be transformed with Abelson's mouse leukemia virus (MuLV). Also in this case there is an unfavorable fixation and a very poor production of antibodies. .

2. Long-term cultures of untransformed B-lymphocytes

The latest results, described in Špredni B. et al. · Long-term culture and cloning of nontransformed human B-lymphocytes. J. Exp. Copper. 154, 1500-1516 (1981), Howard, 'M. and others: Long-therm culture of normal mouse B-lymphocytes. Why. Natl. CAD. Sci. USA in press) show the possibility of culturing and cloning lymphocytes permanently without transformation while maintaining special growing conditions, for example permanent mitogenic stimulation of lymphokine-containing prosCS 276 448 B6 4 classes and the like. These methods are not yet suitable for the conventional production of monoclonal antibodies.

The prior art can therefore be summarized as follows:

B-lymphocytes from normal donors can be artificially immortalized. The hybridoma formation technique uses live myeloma cells, which multiply indefinitely in culture for fusion with antigen-stimulated B cells. The hybrid cells thus obtained are isolated by HAT selection and cloned into individual cultures. Hybridoma clones that produce antibodies with the desired specificity are then propagated to produce a monoclonal antibody. So far, HAT selection has significant disadvantages due to chromosome losses and the formation of hybrid immunoglobulins.

Alternatively, B cells can be infected with specific viruses to produce malignant cells that grow steadily to obtain antibodies. However, these cells produce only small amounts of antibodies. .

Due to the greater production of monoclonal antibodies with defined antigen binding specificity, the hybridoma technique is by far the most preferred.

However, the hybridoma technique also has major disadvantages. The most serious disadvantage is that the cells used for fusion are limited to several species, especially lymphocytes and nerve cells.

The object of the invention is to obviate these disadvantages and to propose a new, advantageous method for the production of sustainable cell lines of animal and human origin.

The present invention relates to a process for the production of permanent cell lines of animal and human origin by fusion of normal cells of human and animal origin with biological components which affect the sustainability of culture in vitro, characterized in that it is subjected to fusion of normal human or animal cells with karyoplasts, cytoplasts, nuclear or a cytoplasmic fraction rich in mitochondria of lymphatic or epithelial tumor cells which are not themselves capable of proliferation and are derived from transformed cells, the fusion is performed in the presence of polyethylene glycol or Sendai virus, and the obtained cells are grown in a nutrient medium without selection agents.

It is essential for the method according to the invention that the fragments used in the fusion are completely free of reproducible cells and cannot in themselves reproduce.

Surprisingly, in carrying out the method of the invention, the predominance of the normal cell cytoplasm does not exclude the malignant properties of the other partner and therefore eliminate the ability to grow permanently, although it is known that when fused transformed cells with the normal cytoplasm in Shay WJ et al .: Suppression of tumorigenicity in Cybrids.

It was also not expected that cell nuclei would associate with isolated nuclei of, for example, myeloma cells on a common genome if the myeloma cytoplasm was not transferred to the hybrid cell.

The fusion is carried out in a known manner, preferably in the presence of polyethylene glycol or Sendai virus, since in this case a higher proportion of fusion can be achieved, as in the case of hybridomas. Other known means to facilitate this procedure are also useful.

Fragments of transformed cells can be obtained, for example, from myeloma cells in a known manner. Preferably, the cell wall is disrupted, for example, after centrifugation, the nuclear fractions are removed from the cytoplasm fraction and the fractions are used individually. Cell disruption is preferably performed by swelling glycerol and transferring to glycerol-free buffer. Another preferred method is to produce caryoplasts and cytoplasts by treating the cells with cytochalasin B, an antibiotic that is commercially available. The procedure is known from Biochem. Biophys. Res. Comm. 63, 669-674 (1975). In this process, for example, only a cell nucleus surrounded by a membrane, as well as karyoplasts and cytoplasts can be obtained. Kernels or can be used

CS 276 448 B6 fractions of the cytoplasm that are no longer surrounded by the cell membrane. Mechanical cell crushing, which can also be used, is known and needs no explanation.

The cell fragments can be used fresh immediately after recovery or later, in which case they are preserved by lyophilization.

Transformed cells are those cells that do not respond to normal growth conditions in vitro and in vivo. Examples are malignant cells, for example cancer cells, cells altered by virus injection (e.g. Eppstein Barr) and cells altered by other carcerogenic substances.

If cell fractions are used, these fractions may not be completely pure, but must not contain intact proliferating cells.

An essential feature of the method according to the invention is the fact that the hybrids obtained are not exposed to competition from non-hybrid, in vitro cultivable cells and that thus the growth of such cells does not have to be suppressed, for example by HAT selection. In this way, it is also possible to eliminate the very unfavorable influence of the selection environment on the hybrids and thus to improve the yield and the viability of the permanently cultivable cells.

Furthermore, it is no longer necessary to use cells sensitive to the HAT environment in the formation of hybrids. A variety of sustained growing cell lines can be obtained which do not have this property and would not be useful in conventional hybridoma techniques. However, the fragments can be used for fusion in carrying out the method of the invention.

The cells can preferably be treated with cytochalasin B, thereby pushing the nucleus to an extreme position. Then, for example, a cell plasma free of nuclei and a nucleus surrounded by a cell membrane and a small amount of cytoplasm (caryoplast or minicell) can be obtained by centrifugation. Both the karyoplast and the cytoplast are no longer able to multiply, but they retain their specific functions for several hours to days. This procedure requires a relatively high concentration of cytochalasin B and is fully reversible. Smaller concentrations suppress cell division without, however, displacing the nucleus. A standard method for producing cytoplast and caryoplast for free cells has been described in Wigler®. N. a Weinstein I. B.s Preparative methods for obtaining enucleated mammalian cells Biochem. Biophys. Res. Comm. 63, 669-674 (1975).

It has been shown that not only B-lymphocytes can be used in carrying out the method according to the invention, but also a number of other cells of animal and human origin, which can also be immortalized, as can be seen from Examples 6 to 8. These are mainly different cell types such as T -lymphocytes, which transmit cellular immunity and regulate the immune system, endothelial cells, i.e. cells from the human venous wall and melanoma cells, isolated from tumor metastases.

The method according to the invention makes it possible to transfer a number of cells to a permanent culture and thus also solves the problem of producing antibodies, enzymes, clotting factors and similar cells synthesized by the cells in vitro. The cultures obtained by the process according to the invention make it possible to replace experimental animals in certain areas even when testing chemicals.

The cell lines obtained can thus be used to obtain cell products, for example, monoclonal antibodies, clotting factors, lymphokines, enzymes and in particular proteins and other products.

Of particular importance is the production of monoclonal antibodies by permanently cultivating lymphocytes, endothelial cells, melanoma, liver cells, kidney cells, by obtaining clotting factors from T-lymphocytes + B-lymphocytes and / or macrophages and by obtaining lymphokines from glandular cells, as well as hormones and the like. It is clear that still other interesting cell products can be obtained by the method according to the invention.

. The recovery of cell products is not limited to the starting cells. Permanently growable cell lines can also be used to express cell products that were not produced by the original cell and that are heterologous by passing on genetic information by obtaining a new combination of genes in a hybrid cell. This can be done, for example, by transformation. Experiments have shown that the permanently cultured cell obtained by the method according to the invention is possible by means of a vector

CS 276 448 B6 6 transfer information and achieve expression of the product for the production of which the vector encoded.

Permanently cultivable cells can also be used as test objects for various active substances. They can also be sources of genetic information by isolating the part of the hybrid cell that carries the genetic information, i.e. the genome, part of the genome or RNA, and then transforms it into a suitable microorganism in a known manner. From which the desired cell product is then obtained. .

In this way, cell products, such as monoclonal antibodies and other cell products, can be obtained by not using the resulting hybrid cell directly to produce the cell product, but only to transfer the genome, part of the genome or RNA to a microorganism, which is then used to obtaining a monoclonal antibody or other cell-produced substance. In this embodiment of the method of the invention, the genome of the hybrid cell is isolated in a known manner and transformed with a suitable vector, using known vectors and standard methods using a suitable microorganism.

The transformed microorganism is then grown in a conventional manner to obtain the desired cell product. These are mainly E. coli strains.

The invention will be illustrated by the following examples, in which the following abbreviations will be used

and trade names: AK antibody and immunoglobulin chain H and L heavy and light chain Ig molecules Land 2,6,10,10,14-tetramethylpentadecane HAT-selection environment containing hypoxanthine, aminopterin and thyme midin TK thymidine kinase HGPRT hypoxanthine-guanine phosphoribosytransferase EBV Epstein-Barr virus MuLV Abelson's mouse leukemia virus CB cytochalasin 8 antibiotic (ALDRICH BIOCHEMICALS Milwaukee USA) DMSO dimethyl sulfoxide ABOUT MY Dulbecco's minimal basic environment FKS fetal calf serum Ficoll polymer cane sugar (PHARMACIA) PEG polyethylene glycol PBS sodium chloride, phosphate buffered UNDER peroxidase ABTS ammonium salt of 2,2'-azinodi (3-ethylbenzothiazole -6-sulfonic) EBSS Earle's balanced soy solution RPMI 1640 Rosewell Park Memory Institute (nutrient environment) Tris tris (hydroxymethyDaminomethane PBL peripheral blood lymphocytes MNC mononuclear cells (lymphocytes, monocytes) hTSH human thyroid stimulating hormone

7 CS 276 448 B6 / 3 -hTSH β -chain of said hormone FA Freund's auxiliary reagent CFA complete Freund's auxiliary reagent IFA Methocel 1500 incomplete Freund's adjuvant methylcellulose (FLUKA) CMV cytoplasmic membrane FITC-Covaspheres fluorescein isothiocyanate in the form of beads (COVALENT TECHNICALS Ann Arbor, Mich. USA) EAZ Ehrlich's carcinoma cells (ATCC, CCL 77)

Example 1

A Production of karyoplasts and cytoplasts from mouse myeloma cells line P3X63 Ag 8.653 ATCC No-CRL-1580 by the method of Wigler, Μ. H. and Weinstein, I. B .: A preparative method for obtaining enucleated mammalian cells. Biochem. Biophys. Res. Comm. 63, 669-674 (1975).

A.l Materials

Cytochalasin B (CB, Aldrich Biochemicals, Milwaukee, USA) was dissolved in dimethyl sulfoxide (DMSO Merck) at a concentration of 2 mg / ml and stored as a stock solution at 4 ° C.

Ficoll-400 (Pharmacia, polymerized cane sugar) is dissolved in redistilled water at a concentration of 1 g / ml, sterilized in an autoclave and used as a 50¾ stock solution, stored at -20 ° C.

Dulbecco's minimal basal medium (DMEM) at single and double concentrations, contains fetal calf serum, (FKS), L-glutamine at a concentration of 200 mmol / liter, streptomycin and penicillin (Boehringer Mannheim). Cellulose nitrate tubes are sterilized by ultraviolet radiation.

Ag 8.653 ATCC CRL-15B0 Myeloma Cells: The line is described in Kearney, ΰ. F. and others. A new mouse myeloma cell line that hat lost immunoglobulin expression but permits the construction of antibodysecreting hybrid cell lines. J. Immunol. 123, 1548-1550 (1979). The line is resistant to acaquinine, sensitive to HAT, and does not synthesize either the H chain or the L chain of immunoglobulin Ig. Maintained in DMEM + 15% FKS + glutamine + penicillin-streptomycin + pyruvic acid (full DMEM medium at 37 ° C in an atmosphere with 7% carbon dioxide.

A.2 Methods'> 3

Enucleation: 10 x 10 6 Ag 8 ^ 653 cells are centrifuged for 5 minutes at 10 rpm and the cells are resuspended in 12 ml of 12.5 F Ficoll-DMEM-CB-DMSO solution until a cell-free suspension is formed. Each roll of this suspension is overlaid with a pre-prepared Ficoll gradient for 12 hours and overlaid with 2 ml of Ficoll-free OMEM-CB-DMSO solution. Tubes with this gradient are centrifuged in an ultracentrifuge for 60 minutes at 25,000 rpm and 31 ° C.

At the end of the blasting, the macroscopically visible fractions (bands) are collected separately from above using a long cannula syringe, diluted in each case with 20 ml of DMEM medium without additives, centrifuged again and resuspended in fresh DMEM.

The following 4 fractions are obtained. .

a) Cell debris, at the interface 0 to 12.5 F of Ficoll,

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b) nuclear-free cytoplasts in the region of 15 to 16% of Ficoll,

c) nuclei without detectable plasma and approximately 2% non-nuclear cells in the range of 17 to 25% of Flcoll,

d) according to the morphological criteria of an intact cell with a nucleus and well-distinguishable plasma, the fraction contains a small amount of nuclei without plasma as sediment at the bottom of the tube.

When counting cells, it could be shown that 8 x 10 ⁷ Ag of 8.653-cells were contained in fraction b), 1.25 χ 10 ⁶ cytoplasts in fraction c) 4 χ 10 ^é caryoplasts and in fraction d) 1.1 χ 10 ⁷ intact cells.

B Fusion of mouse spleen cells with isolated myeloma karyoplasts, cytoplasts and sediment cells from Experiment A.

B.l Material.

Fusion reagent: 20 g of polyethylene glycol (PEG-4000) are melted in an autoclave, cooled to 56 DEG C. and mixed at this temperature with 20 ml of DMEM.

HAT-selection medium: 4 χ 10 ^-7 M aminopterin, -4-5 x 10 M thymidine and 3.1 x 10 M hypoxanthine are added to the full OMEM medium.

Growing vessels: tissue culture vessels (Cluster 24 and Cluster 96, Costar, Cambridge, Mass., USA.

B.2 Methods

Fusion: fractions b), c) and d), prepared in experiment A, are separately mixed with spleen cells in a ratio of 10: 1 and sediment is formed by centrifugation. The supernatant is carefully separated. 0.8 ml of a 50% PEG solution was added to the sediment at 37 ° C for 1 minute, the mixture was stirred with gentle constant shaking, then 5 ml of DMEM was added over 5 minutes at room temperature. After addition of another 20 ml of DMEM medium, the cells are allowed to settle, resuspended in 5 ml of fresh whole OMEM medium and divided into 10 tissue cultures (Costar) overlaid with abdominal macrophages. On days 1, 2, 3, 5, 7, 10 and 13, full DMEM medium is added to each culture.

Abdominal macrophages as feeder cells: the day before fusion, Balb / c mice are sacrificed under sterile conditions, 4 to 5 ml of PBS are injected into the abdominal cavity, and after 1 minute, this material is aspirated again. The washed cells are washed with DMEM. The complete medium is adjusted to a cell density of 2 χ 10 ⁵ and dispensed in 0.5 ml portions into cell culture vessels (Costar).

Spleen cells: The spleen is removed from the Balb / c mice immediately before the phase under aseptic conditions and its cells are suspended in DMEM. The cell clumps and tissue sections are filtered through a layer of mule.

Mouse immunoglobulin ELISA: microtiter plates are overlaid with mouse Ig antibodies from sheep (IgG fraction: 10 micrograms / ml 0.9% sodium chloride, 150 microliters of antibody solution per well). 100 microliters of supernatant are pipetted into each well and the plates are incubated for one hour at room temperature. After aspirating the supernatant and washing twice, the chambers are overlaid with 100 microliters of a solution of Ig-POD conjugate against mouse serum covalently bound with sea onion peroxidase, and the mixture is incubated for one hour at room temperature. After washing three times, 100 microliters of substrate solution (ABTS) is pipetted into each chamber and the color is determined photometrically.

B.3 Results

The cytoplast, caryoplast, and sediment fractions prepared according to Experiment A were fused in parallel with the spleen cells of Balb / c mice and divided into 10 1 ml cultures.

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In each case 5 cultures are grown without HAT (plate I) and 5 cultures with HAT (plate II).

Up to day 21 after fusion (n. F.), no growth of lymphoid cells was observed macroscopically or microscopically in any chamber, except for chambers 4A, 4B, 40, 30 and 3D on plate I, which contained sediment material without HAT medium after fusion. Colonies that grew rapidly could be observed in these chambers as early as 5 days after fusion. On day 8 after fusion, HAT medium was added to these chambers. Within 4 days, all colonies died.

On day 27 after fusion, it was possible to observe first in some, later in almost all chambers of the colony, which were formed by large spherical, transparent cells that did not adhere to the substrate. On day 65 after fusion, with the exception of chambers I-3B, II-1A and II-4A, all chambers were filled with lymphoid cell colonies. Examination of the supernatant for mouse Ig at this time showed positive to strongly positive ELISA values in Table I in all subcultures except the above-mentioned chambers, which did not develop cultures.

Table 1

ELISA to detect mouse immunoglobulin in the supernatant of Experiment B (day 65 after fusion).

Plate I with chambers: without HAT (Exceptions: 4A, 4B, 4C, 3C, 3D: + HAT, day 8 to 15)

1 2 3 4 5 6 AND 664 601 706 632 B 526 766 011 633 623 C 576 769 855 D 794 1500 791

= post-fusion cytoplasts = post-fusion caryoplasts = post-fusion sediment cells

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Plate II with chambers: with HAT (day 1 to 14)

1 2 3 4 5 6 AND 008 568 580 000 B 267 538 539 547 C 656 530 729 822 0 848 570 605

3 negative-Ktr. 1 (DMEM - full environment): '010 negative-Ktr. 2 (DMEM, without FKS): '040 positive-Ktr. 1 (mouse serum 1 x 10 ^-3 ): '723 positive-Ktr. 2 (mouse serum 1 x 10 ~ ² ):> 1500

a) Fusion of karyoplasts with mouse spleen cells resulted in cells that could be propagated in vitro and secrete immunoglobulin. Although only a small amount of the cytoplasm of malignant cells was transferred to the hybrid, the properties of permanent growth could not be ruled out.

b) Karyoplast-induced hybrid cells produced and secreted murine immunoglobulin in an amount corresponding to the hybridoma. This means that the lack of the proportion of cytoplasm Ag 8.653 did not affect the ability to produce and secrete a hybrid of karyoplasts and spleen cells.

c) The fusion of cytoplasts and spleen cells resulted in cell clones that propagated in vitro to produce antibodies. A satisfactory explanation for this surprising phenomenon is not yet available.

Example 2

Fragmentation of cells by glycerol disruption and fusion with human blood lymphocytes

Material ’>

Earl's balanced salt medium (EBSS), APMI 1640 medium, fetal calf serum (FKS) (Boehringer Mannheim), 8-azaguanine (8-Ag) (Serva Heidelberg), agar (Bacto-agar 1614) (Difco, company Hedinger KG, Stuttgart). The HS SULTAN ATCC CRL-1484 plasmacytoma line was thawed as freeze-preserved cell material according to ATCC instructions and grown in culture.

According to the method described in Proc. Natl. Acad. Sci, USA. 71, 2679-2683 (1974), 8 x 10 ⁷ HS Sultan cells are grown in 100 ml of complete RPMI 1640 medium containing 20 micromoles of 8-Ag, the culture is grown for 48 hours. Surviving cells are further grown in 10 ml of full RPMI 1640 medium without 8-Ag for a total of 10 days. The cells are then transferred to agar plates Coffino, P. et al., Proc. Nati. Acad. Sci. USA. £ 8, 219-223 (1971), supplemented with RPMI 1640 + 20 micromoles of 8-Ag, approximately 500 cells per petri dish were used, and the culture was grown in an incubator in carbon dioxide. After 9 days, isolated growing colonies are sterile removed from the agar surface and propagated in full RPMI 1640 medium supplemented with 8-Ag. A clone that was designated HS

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-SULTAN-8Ag-R1, whose cells were doubled every 20 hours, was used for another experiment. These cells were sensitive to HAT. Cells grown at a density of 1 to 5 x 10 6 per ml in HAT medium (RPMI 1640 medium with 0.1 mmol hypoxanthine, 400 mmol aminopterin and 31 micromole thymidine) did not proliferate and completely died within 7 days.

Human peripheral blood lymphocytes (PBL) in 300 ml of venous blood are sterile isolated in a heparin solution at a concentration of 2 units / ml blood and the mononuclear cell fraction (MNC: O lymphocytes, monocytes) is isolated in a standard manner. 3 x 10 MNC are added to a suspension of 100 ml RPMI 1640 + 10% FKS and the mixture is incubated for 24 hours in tissue culture dishes at 37 ° C in a 5% CO 2 medium to separate monocytes.

Methods.

HS-R1 fragmentation: Cells are mixed as described by Jett M. et al .: Isolation and characterization of plasma membranes and intact nuclei from lymphoid cells. 3. Biol. Chern. 252, 2134-2142 (1977) and disrupted by incubation in 10 mmol of Tris-buffer with hydrochloric acid, the nuclei were separated by centrifugation at 200 g for 10 minutes at 4 DEG C. from the remnants of the membranes, which were subjected to sedimentation by centrifugation at 5,000 g. minutes at 4 ° C.

O

Fusion: Approximately 1 x 10 729 HS-R1 nuclei were suspended in human lymphocytes in RPMI 1640 medium in a ratio of 1: 1 and then fused with PEG as described in Example 1 8.2.

O

The remainder of the rest of the cell membranes from approximately 1 x 10 5 HS-R1 cells is overlaid with a suspension of 1 x 10 ⁷ human lymphocytes and fused with PEG.

In a control experiment, mix 2 x 10? HS-R1 cores in full RPMI 1640-free medium and the material is grown on 4 24-well plates (Costar) in a carbon dioxide incubator.

All post-fusion and control cultures were grown in mouse abdominal macrophage plates as feeder cells as described in Example 1.

Monitoring of human Ig in the culture supernatant: Microtitration by ELISA is performed as in Example 1 B.2. Sheep Ig against human serum, purified by immunoadsorptive method, is used for overlaying. The same AK compositions were used to obtain anti-human serum Ig-POD conjugate. Sheep AK reacts with every human Ig and no cross-reactivity with bovine or rat Ig can be demonstrated. ·

Results

Fragmentation with a mixture of glycerol, Tris-buffer and hydrochloric acid: In this way, HS-R1 cells decompose into a nuclear-containing fraction that settles at 200 g and a non-nuclear cytoplasmic fraction and a membrane that settles at 5,000 g. in none of these fractions intact HS-R1 cells. The nuclei were surrounded by more or less unevenly bounded cytoplasm. The yield of the cores was approximately 85%. The fraction containing cell membrane fragments contained, in addition to a small amount of membrane pulp of 0.5 to 2 micrometers in size, without detectable nucleus components. '

A culture containing 2 x 10 ⁷ nuclei in full RPMI medium without HAT did not lead to any growth of HS-RI cells during the observation period of 12 weeks.

_r Culture after fusion: Lymphocytes with nuclei and lymphocytes with cytoplasmic membrane after fusion were divided into 10 plates with 24 cells (Costar) and in each half half of the cells were grown without HAT and half with HAT supplemented in RPMI medium with mouse macrophages. In the second week after fusion, colonies of lymphoid cells can be observed, which gradually grow.

Human immunoglobulin production: On day 21 after fusion, supernatants were examined for content

CS 276 448 86 human immunoglobulin (on day 19, nutrients were obtained in this way, are reported in

Table 2a

ELISA to detect human immunoglobulin in

I + HAT environment completely replaced). The results of the following Tables 2a and 2b.

supernatants (caryoplasts, 21 days after fusion).

I - HAT

1 2 3 4 5 6 AND 029 147 286 147 228 270 B 171 086 050 144 846 121 C 118 156 250 082 179 059 0 120 1148 024 096 023 151

1 2 3 4 5 6 AND 660 233 175 270 410 322 B ' 103 264 229 253 271 260 C 045 343 370 128 150 126 D 171 173 141 116 218 699

II + HAT

1 2 3 4 5 6 AND 135 290 107 279 530 674 8 116 500 469 315 315 627 C 120 050 068 159 226 145 D 191 078 686 110 242 561

II - HAT

1 2 3 4 5 6 AND 381 235 489 514 608 217 B 348 239 214 158 367 163 C 185 275 235 234 322 316 0 219 202 292 283 220 052

Table 2b

Cytoplasts, 21 days after fusion

1 2 3 4 5 6 AND 052 627 917 B 041 326 715 C 071 400 826 0889 0 079 917 494 1016

t T

2 = control culture with nuclei = with HAT = without HAT. In case extinction values 0 to 99 are reported as negative to doubtful, values 100 to 200 as positive and values higher than 200 as strongly positive, the following distribution may be given for cultures obtained by fusion of nuclei.

13 CS 276 448 86 Cultivated hybrids of lymphocytes with nuclei ELISA for human immunoglobulin negative positive strongly positive with HAT n 11 18 19 (n = 48) (¾) (23) (37) (40) without HAT n 2 12 34 (n = 48) (¾) (4) (25) (71)

The following conclusions can be drawn from the results:

Post-fusion cultures obtainable using disrupted cell fractions can be further grown without HAT selection. This cultivation is far less strenuous than preparing cultures with cytochalasin B and provides a material capable of fusing in high yield. A pure division into the nucleus fraction and the cytoplasmic membrane fraction is not achieved in either way. The fraction, which contains predominantly nuclei, and the fraction, which contains predominantly cytoplasm and cell membrane, yield, after fusion with human lymphocytes from the blood, in vitro cell clones capable of proliferating and producing AK. Parallel cultivation of hybrids of lymphocytes and nuclei with and without the addition of HAT shows a negative effect of the selection environment. With the addition of HAT, 23% of primary cultures are Ig-negative and only 40% strongly positive. Without HAT, over 70% of cultures are strongly positive and only 4% are Ig-negative.

Example 3

Fusion of spleen cells from immunized mice with native and fragmented Ag 8.653 myeloma cells

Material

Human thyroid stimulating hormone (hTSH) and its isolated β-chain (j-hTSH), (Boehringer Mannheim). In addition, complete and incomplete Freund's auxiliary reagent (CFA, IFA) (Oifco, Methocel 1500, Fluka) and FITS-Covaspheres (Covalent Tech. Co., Ann. Arbor, Michigan, USA) were used.

Methods

Immunization; Balb / c mice are primarily immunized on day 1 with β-hTSH, 40 micrograms in CFA intraperitoneally, and on day 196 the immune response is enhanced by administration of hTSH, 50 micrograms in IFA intraperitoneally, on day 266 hTSH without vehicle intraperitoneally, and on day 294 administration of hTSH intravenously. in R

Spleen cells at approximately 1 x 10 6 were harvested from immunized mice 3 days after the last injection according to the method of Example 1, B.2 and used for two fusions. 7 7

A 1: 5 x 10 6 spleen cells and 1 x 10 6 Ag 8.653 fusion were mixed as described in Example 1 8.2 and the fusion material was grown in 48 24-well plates containing DMEM in full medium supplemented with HAT and mouse macrophages. ’

The fusion of 2: 1 x 10 6 Ag 8.653 cells was disrupted as in Example 2 by sequential treatment with glycerol and 10 mmol Tris-buffer with hydrochloric acid. The cytoplasm and CMV cell membrane fractions were pelleted at 5,000 g for 40 minutes at 4 ° C. The nuclear fraction was mixed with 7 x 10 ⁷ spleen cells, centrifuged to the CMV sediment, and fused in a standard manner using PEG as in Example 1 B.2, Material thus obtained

CS 276 448 B6 is grown in full DMEM medium in 24 24-well plates with HAT-free macrophage.

Antigen Labeling and Cloning of Hybrid Cells: (FITC-) Covaspheres are overlaid covalently with hTSH (TSH-CS) as recommended by the manufacturer and stored in 5% sodium azide solution. As described in Parks 0.R. et al., Proc. Natl. Acad. Sci. U.S. Pat.

ELISA for TSH-specific antibodies: Overlaying, incubation of culture supernatant, substrate reaction and evaluation are the same as in Example 1 B.2. TSH-POD conjugate was used instead of Ig-POO against mouse serum. The positive control is the serum of hTSH-hyperimmunized mice at a dilution of 1Ό ^- · ⁵ , the negative control is a clone that produces antibodies against unused antigen (mouse antibody against digoxin).

Results

Both in cultures from fusion 1 with intact Ag 8.653 cells and in cultures from fusion 2 with fragments of Ag 8.653 cells, a colony of large lymphoid cells can be observed on day 14 in all wells. The ELISA assay performed on day 14 culture supernatant for antibodies specific for TSH gave results, which are summarized in Table 3 below. Antibodies to TSH could be detected in all subcultures.

Table 3

ELISA to detect anti-TSH in the supernatant, day 14 after fusion, a) fusion 1 (intact Ag 8.653), b) fusion 2 (Ag653-fragments)

a) b)

1 2 3 4 5 6 AND 235 236 212 149 119 212 B 109 221 119 104 176 068 C 116 108 135 139 093 135 D 171 128 120 228 137 145

1 2 3 4 5 6 AND 217 268 267 286 194 291 B 288 278 362 317 280 305 C 207 . 292 252 226 292 271 0 295 376 370 338 310 333

Negative-control (DMEM-full environment): 000

Positive-control (anti-TSH-mouse serum): 362

On day 15, cells that do not adhere to the support from individual wells after fusion 1 and 2 are washed and specifically labeled with TSH antigen after individual batches. This reaction is based on the fact that hybridoma cells, like B-lymphocytes, generally carry a certain proportion of synthesized antibodies in the cell membrane, with antigen binding sites pointing outwards, so that this can be used to differentiate individual clones and cultured.

Example 4

Fusion of human PBLs with intact and fragmented Ag 8.653 cells

Material

Inactivated hepatitis B surface antigen (HB ^ Biotest) is purified from serum protein residues by immunoadsorption. ELISA to detect human _HB -protilátek: Microtiter plates

CS 276 448 B6 is overlaid with purified HB _gl , using 20 micrograms / ml in 0.9% sodium chloride solution. Incubation of the culture supernatant, reaction of the conjugate and substrate and evaluation are the same as in Example 1. B.2.- Instead of Ig-POD against mouse serum, Tg-POD against human sheep serum is used.

Execution

HPBL from a high titer anti-HB _g donor is isolated from 200 ml of venous blood by Ficoll gradient centrifugation. To enrich B cells, T cells are seeded in a standard manner with sheep erythrocytes and then the fractions are separated by a second centrifugation in a Ficoll gradient. The non-rosette cell fraction (HPBL (B)) was grown at a density of 5 x 10 6 cells in RPMI 1640 medium supplemented with 10% autologous plasma, inactivated by heating for 30 minutes at 53 ° C, culturing with 10 micrograms HB _gi in incubator. in the presence of carbon dioxide, change the medium every 12 hours.

Fusion 1: 1 x 10? pretreated HPBL cells (B) are mixed with six 10? of Ag 8.653 cells and the fusion is performed in the presence of PEG as in Example 1 B.2. The material obtained by fusion is grown in RPMI 1640 medium with the addition of 10 ¾ human plasma and HAT on 4 24-well plates (Costar).

7

Fusion 2: 1.1 x 10 6 Ag 8.653 cells are fragmented by glycerol. 1 x 10 6 8.653 Ag 8.653 nuclei are mixed with six 10 6 HPBL (B) and overlaid on cytoplasmic sediment and cell memO gate from 1 x 10 6 8.653 cells. After PEG fusion, the material was grown in 4 24-well plates in RPMI 1640 medium with 10 ¾ human plasma without HAT.

Results

In all wells after fusion 1 and 2, large adherent cells grow strongly on day 3. On day 5, non-adherent cells are carefully resuspended and divided into 2 new 24-well plates. (For example, Al, B1 and Cl). On day 28, small colonies in A2 and A3 can be observed in the fusion material 1, only adherent cells in the remaining chambers, and a massive growth of multiple colonies in all chambers except C3 in the fusion material 2. On day 35, an ELISA was performed to detect human immunoglobulins specific for the hepatitis B antigen, the results of which are summarized in Table 4 below. 5 of the 12 cultures after fusion 2, i.e. fragments of Ag 8.653 cells, the culture in normal medium was clearly anti-HB _g- positive.

Example 5

Fusion of HPBL with fragments of the HS SULTAN line of human plasmacytoma

Material '

HS SULTAN cells were obtained from the American Type Culture Collection (ATCC) under the designation CRL-1484 as a cryopreserved cell material and thawed according to ATCC recommendations and transferred to culture.

Execution.

HPBL was obtained as in Example 4 from the same donor and in the same treatment.

Fusion: 4 x 10 6 HS SULTAN cells were disrupted by glycerin. The cytoplasm and membrane fractions were sedimented at 5,500 g for 40 minutes and overlaid with a 4 x 10 6 mixture. cores HS SULTAN and 4 x 10? HPBL (B). The PEG fusion was performed according to Example 4, the material thus obtained was grown in 12 24-well plates (Costar) in RPMI 1640 + medium.

CS 276 448 B6 + 20% FKS + pyruvic acid + insulin (Novo 2 units / ml) + 1% non-essential amino acids (BM) + 1% Methocel 1500 without HAT.

Results of days after fusion, growth of large, non-adherent lymphoid cell colonies can be observed.

Example 6

Immortalization of human T-lymphocytes

6.1 Material and methods

T cells are isolated in a known manner by plating with sheep red blood cells followed by Ficoll gradient centrifugation from the lymphocyte fraction and further treated with buS immediately or after three days of culture. Ehrlich ascites tumor cells (ATCC, CC1 77) are grown in DMEM medium with 10% horse serum and donor fragments for transformation. Fragmentation of this tumor (EAZ) cells is performed according to the method of 3ett et al. (3. Biol. Chem. 252, 2134-2142 (1977)) by the action of glycerol. The fraction, which contains predominantly nuclei, is separated by centrifugation and discarded. The mitochondrial-rich (CMV) -rich fraction containing cytoplasmic membrane is used for transformation. 5 x 10? T cells are overlaid with excess PHA-lectin (Difco), mixed with the CMV fraction from EAZ and incubated for 20 minutes at room temperature. Sedimentation of the mixture is achieved by centrifugation, the liquid supernatant is completely removed and replaced by 1 ml of 50% PEG solution. After 1 minute of treatment, the PEG solution is diluted by adding RPMI 1640 medium and then separated from the cells by centrifugation. The cells are mixed with RPMI medium with 20% fetal calf serum (FKS, BM), divided into 12 well plates and grown at 37 ° C in an atmosphere of 5% carbon dioxide. The characterization of the cells according to the surface properties is performed by roseting with the action of sheep red blood cells according to the method of Kaplan, Μ. E. et al. 3. Immunol. Methods, E, 131 (1974) and immunofluorescence using both T-cell specific OKT-3 antibodies (Ortho, Reinherz EL et al. 3. Immunol. 123, 1312 (1979)) or MAK 4-11 (Rieber P. et al., Hybridoma (59 (1981)), and fluorescence-labeled immunoglobulin against human serum (Ig, Dako) to detect Ig membranes typical of B-cells.

6.2 Results

During the first 14 days, most cells in the cultures die. On day 21, colonies of small to medium-sized cells can be observed in the cell debris, which multiply continuously. Colony growth can be observed in all chambers up to 50 colonies in one chamber. On day 30, kolothe colonies are transferred to larger vessels and further propagated. The cells are analyzed on the basis of surface properties, the following results were obtained:

and) material positive on E-rosettes > 95 q, 'i b) material positive on OKT-3 > 90 * 0 C) material positive on 4-11 > 90 9, * 9 d) material positive on ^l9 s < 5 O, * 0

6.3 Note

Fragments obtainable from EAZ (permanent mouse lines) by the action of glycerol, when fused by the action of PEG with isolated T-lymphocytes, give cells which grow steadily in culture and which can be unambiguously identified on the basis of surface properties as T-lymphocytes.

CS 276 448 86

Example 7

Immortalization of human endothelial cells

7.1 Material and methods

All growing containers are covered with gelatin. The culture supernatant from a mixture of RPMI 1640 medium and medium 199 (BM) with 20% FKS in a ratio of 1: 1. Human endothelial cells are obtained according to the method of Jaffe et al., J. Clin. Invest. 52, 2745-2756 (1973) by treatment with a solution of collagenase (Difco) from fresh umbilical cords and the primary culture is grown for approximately 14 days before transformation. Adherently growing endothelial cells are used for transformation, which are released by treatment with a solution of trypsin and ethylenediaminetetraacetic acid (BM) and fused in suspension as described in 6.1 with the CMV fraction from EAZ. The cells thus treated were further grown at an initial concentration of 5 x 10 ⁵ per 75 ml flask in a carbon dioxide incubator. The control is always the proportion of endothelial cells from the primary culture without the CMV fraction with the addition of PEG and with inoculation. Once the cells form a continuous surface at the bottom of the vessel, they are separated by trypsin and EDTA and transferred to a fresh vessel in a ratio of 1; 3 regular passages.

7.2 Results

Endothelial cells, after fusion with the CMV fraction, adhere to the bottoms of the vessel, usually at least 20 to 30% and form a continuous cell layer within 2 to 3 days. Control cells proliferate poorly and usually die without forming a continuous cell layer within 21 days. PEG-free endothelial cells proliferate to a maximum of the third passage, then growth is stopped and the cells dissolve at the bottom of the vessel. In 8 different samples of endothelial cells, which were taken from a total of 18 umbilical cords, the cells could be grown after CMV fusion without any problems for more than 10 passages. Transformed endothelial cells can also be stored without loss of viability and ability to proliferate in liquid nitrogen.

7.3 Notes

Human umbilical cord endothelial cells cannot be cultured for more than 3 passages without transformation by the method of the invention, as also described in the literature, for example in Grimbrone M. A. Progress in Haemostasis and Thrombosis, Vol. 3, Maciag, T. et al., J. Cell Biol. 91, 420-426 (1981). Fusion by the mitochondrial-rich CMV fraction from EAZ alters the properties of endothelial cells in culture so that they can be easily transferred through the critical third passage and further propagated, currently the cells are in passage 23 without fatigue. culture.

Example 8 '

Immortalization of human melanoma cells

8.1 Material and methods

Tissue containing melanoma cells was obtained by surgically removing a lymph node metastasis under sterile conditions, divided into small aliquots, and stored in liquid nitrogen until fusion. The CMV fraction from EAZ was obtained as in 6.1,

Prior to fusion, the melanoma cell-containing material was thawed and a single cell suspension was obtained by trypsinization. The fraction of large, brownish-red pigmented melanoma cells was stripped of the Ficoll ballast lymphocyte fragment by centrifugation and fused. Fusion is performed as described in Example 6.1 with the CMV fraction in the presence of PEG used was approximately 4 x 10 ⁵ melanoma cells and CMV 1 x 10 ^é EAZ. Controls were 4 x 10 ^{5 cells}

CS 276 448 B6 18 melanoma, which were further grown in the presence of PEG without fusion with CMV. After fusion, the cells were divided at a density of 1 x 10 6 into chambers containing RPMI 1640 + 20% FKS and grown at 37 ° C in an atmosphere of 5% carbon dioxide.

8.2 Results

In all four subcultures, after fusion of CMV with melanoma cells, colonies of typically pigmented cells grew to day 28 in the form of semiadherent cell clusters, which grew continuously. There was only a slight increase in the control culture, but then from the 8th day the cells were granulated and on the 22nd day only cell debris could be observed in the culture.

8.3 Notes

Human melanoma cells from frozen metastatic tissue could be transformed by fusion from CMV and then obtaining cells growing and proliferating in vitro. Control melanoma cells of the same origin died under the same culture conditions.

Example 9

Introduction of an eukaryotic DNA vector into immortalized human endothelial cells

9.1 Detection of transferred material in cells in Hirt's supernatant

9.1.1 Material

Plasmid Z-pBR322 / Rchr βG-A 425 B (Dierks, P. et al., Proc. Natl. Acad. Sci. USA 78, 1411-1415 (1981) contains the 2,070 base pairs (Bp) fragment of the rabbit β-globin gene). .

The Cla-Pvu I fragment of plasmid pBR322 was replaced with a 3.039 Bp HPa Ι-Bam Η 1 fragment containing the entire region of the previous gene and the origin of the SV 40 gene region (Tooze, 3 (1980) in DNA Tumor Viruses, Harbor Laboratory and B. Wiering). and others, Cetus-UCLA Symposium on Gene regulation 1982). This plasmid will be hereinafter referred to as pBR322 Rfa G SV 40.

9.1.2 Methods

Human endothelial cells were immortalized and allowed to grow as in Example 7. 10 ⁿ cells were transformed using 6 micrograms of pBR322 Rfa G SV 40 with 4 micrograms of calf thymus DNA after sonication using the calcium phosphate precipitation method according to publications. Graham FL et al., Virology 52, 456-467 (1973) and Wigler M. et al., Cell 14, 725-731 (1978). 10 hours and 40 hours after transformation, the Hirt supernatant is obtained according to Hirt B., 3. Mol. Biol. 26, 365-369 (1967).

These competitors were separated on a 1% agarose gel and transferred according to Southern E. M., 3. Mol. Biol. 98, 503-517 (1975) on nitrocellulose paper. Plasmid pBR322 R 1 G SV 40 was labeled with P using the translation method of Rigby P. W. et al., 3. Mol. Biol. 113, 237-251 (1977) and subjected to DNA-transferred hybridization on a nitrocellulose filter according to the method of Maniatis T. et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982). The filter is irradiated at -70 ° C, X-ray film is used.

9.1.3 Results

Human endothelial cells transformed with plasmid pBR322 Rg G SV 40 emit a strong hybridization signal that corresponds to the authentic plasmid with respect to agarose gel motility. Comparison of Hirt 's cell supernatant signals 40 hours after transformation and 10

ES 276 448 86 hours after the transformation, it is possible to obtain evidence of a four- to five-fold increase in the intensity of this signal,

Hybridization of DNA that is smaller than the inserted plasmid is also possible. However, no signals could be detected in immortalized human endothelial cells without transformation.

The same distribution of hybridization signal intensity could be demonstrated when HeLa cells were transformed with pBR322 R ^ G SV 40.

9.1.4 Summary

The fact that DNA hybridization can be demonstrated in the Hirt supernatant of human immortalized epithelial cells after transformation with plasmid pBR322 R ^ G SV 40 on an inserted plasmid labeled with ^ ² P, which cannot be detected on cells without transfer, is evidence that the vector was inserted into a eukaryotic cell line. The observation that the hybridization signal after transformation is 4 to 5 times stronger after 40 hours than after 10 hours suggests that the transferred plasmid has replicated in the cells.

9.2 Detection of specific transcripts of rabbit β-globin in cells of immottalized human endothelium, transformed with plasmid pBR322 R 1 G SV 40.

9.2.1 Material

Nuclease S *, polynucleotide kinase and calf intestinal phosphatase are used in the form of conventional means (Boehringer Mannheim).

9.2.2 Methods

Immortalized human epithelial cells are propagated as described in section 9.1.2 and then transformed with a vector obtained from the rabbit globin gene. 48 hours after transformation, 1 to 2 x 10 ⁶ cells were disrupted and RNA was extracted using lithium chloride and urea according to the method of Auffray, 0. et al., Eur. 3. Biochem. 107, 303-314 (1980).

Obtaining a hybridization probe specific for rabbit β-globin.

Plasmid pBR322 R1G SV 40 was digested with the enzyme HaeIII and a fragment comprising +135 to -75 according to Dierks P. et al., Proc. Natl. Acad. Sci. USA 78, 1411-1415 (1981) and Van Oyen A. et al. Science 206, 337-3444 is isolated on a 2% agarose gel and chromatographed on DEAE-cellulose according to the method of Muller W. et al. 3. Mol. Biol. 124, 343-358 (1978). The fragment is dephosphorylated by calf intestinal phosphatase a

P then indicates F ^/ - ³² P-ATP and T ₄ -polynukleotidkinázou caused described by N. Mantei et al Gene £ 0, 1-8 (1980). '

Detection of nuclease

The kinase-treated fragment was co-precipitated with 10 micrograms E. coli T-RNA (Boehringer Mannheim) ethanol and then dissolved in 100 microliters of 0.4 M / L sodium chloride, 1 mM / L ethylenediaminetetraacetic acid, 40 mM / L buffer pH 6.4 and 80% formamide according to the method of Mantei N. et al. Nature 281, 40-46 (1979). 25 micrograms of cellular RNA were dried in vacuo, dissolved in 10 microliters of probe (0.01 pMol 30,000 cpm), denatured as described above, sealed in a glass capillary, and hybridized at 48 ° C for 16 hours. In a control experiment, the probe hybridizes to rabbit globin m-RNA (Miles). The sample is diluted with 100 microliters of 0.2 M / l sodium chloride 50 mM / l sodium acetate pH 4.5 1 mM / l zinc sulfate and 0.5% glycerol and then incubated with 50 units of S. nuclease at 30 ° C. ° C for a total of 60 minutes according to Weaver R. et al., Nucl. Acid. Res. £, 1175-1193 (1979).

CS 276 448 86 20

The samples were treated with phenol, co-precipitated with 10 micrograms of E. coli t-RNA from ethanol and then washed with 80% ethanol for 20 minutes at -70 ° C under vacuum, dissolved in 5 microliters of a color solution containing 0 05% bromophenol blue, 0.05% xylolxyanol, 1 mM / l ethylenediaminetetraacetic acid in 90% v / v formamide, the mixture was heated on a boiling water bath for 2 minutes and then subjected to electrophoresis on a 5% polyacrylamide gel. (89 mM / l Tris buffer, 89 mM / l boric acid, 1 mM / l ethylenediaminetetraacetic acid and 7 M / l urea). The gel was subjected to autoradiography using an X-ray film and a crosslinking shade at -70 ° C (Fuji).

9.2.3 Results

The size of the protected fragment can be determined by comparison with a fragment of known size. (P-labeled plasmid pBR322 x Hinf 1 and pBR322 x Hae III). Untransformed cells do not emit any globin-specific hybridization signals except for a renatured hybridization probe (210 Bp). RNA extracted from immortalized human epithelial cells after transformation contains two fragments of 80 to 90 Bp in size. Similar transcripts have also been demonstrated in Grosveld et al., Nature 295, 12p-126 (1982) and Weidle U. et al., Nature (1981), and have been attributed to the presence of an internal cryptic position in the rabbit β-globin gene. Transcripts whose origin is in the cap position of the rabbit globin gene could not be detected. In a comparative experiment, HeLa cells were transformed with plasmid pBR322 R / JG SV 40. The correct transcripts (135 Bp) and transcripts derived from the presence of an internal cryptic position according to Grosveld G. et al., Nature 295, 120 to 126 (1982) and Weidle Ua et al. Nature (1983). '

9.2.4 Summary

The fact that rabbit β-globin-specific transcripts can be detected in immortalized human epithelial cells that contain a vector derived from SV 40 from the rabbit / S-globin gene indicates that, after transformation, this cell line is suitable for expression of the incorporated cloned genes. .

Claims (4)

PATENT CLAIMS A process for the production of permanent cell lines of animal and human origin by fusing normal cells of human and animal origin with biological components which cause the sustainability of culture in vitro, characterized in that it is subjected to fusion of normal human or animal cells with karyoplasts, cytoplasts, nuclear or a cytoplasmic fraction rich in mitochondria of lymphatic or epithelial tumor cells, which are themselves incapable of proliferation and are derived from transformed cells, the fusion is performed in the presence of polyethylene glycol or Sendai virus, and the obtained cells are grown in a nutrient medium without selection agents. 2. The method of claim 1, wherein the karyoplasts or cytoplasts of the transformed cells are used after treatment with cytochalasin B. 3. The method according to item 1, characterized in that cell fragments, in particular nuclear fractions or cytoplasmic fractions from transformed cells, are used after treatment with a lytic substance or after mechanical crushing of the cells. 4. The method according to items 1 to 3, characterized in that myeloma cells or cells infected with Epstein-Barr virus are used as transformed cells.