RU2164240C2 - Method of cultivation, fibroblasts, method of cell separation - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: cultivation of human stem cells is carried out ex vivo in liquid cultural medium with medium exchange rate 50-100% of every day change at cell density 1 x 10⁴ - 1 x 10⁷/ ml of medium. Cultivation is carried out in the presence of growth factors and/or transformed stromal cells secreting growth factor. Transfer vector of gene providing reading the stable genetic information is added to culturing medium additionally. For separation of neoplastic cells from normal ones hemopoietic cells combined with stromal cells are subjected for liquid flow making shear stress at value 1.0 dyne/cm² or above. Invention can be used for hemopoietic cell cultures growing and to optimize growing stem cells and cell-precursors. EFFECT: improved method of cultivation and separation of cells. 21 cl, 9 dwg, 7 tbl

Description

 The present invention relates to methods, bioreactors and compositions for culturing and transforming mammalian cells in a culture medium, in particular for growing and transforming hematopoietic cell cultures.

 All blood cells in a healthy adult, including red blood cells, white blood cells, platelets and lymphocytes, are produced in the bone marrow as progenitor cells during differentiation. These cells, in turn, are produced from very mature cells called undifferentiated progenitor cells, which, as shown by the analysis, produce mature hemocyte colonies in 1-3-week cultures in semi-solid medium, for example methyl cellulose or agar. Said precursor cells are essentially derived from a class of progenitor cells called stem cells. Stem cells are capable of dividing both self-maintenance and self-differentiation in undifferentiated progenitor cells. Thus, stem cells during division produce both embryoblast cells and somewhat more differentiated progenitor cells. In addition to the production of hemocytes, stem cells can also increase the yield of osteoblasts and osteoclasts, and possibly cells of other tissues. In this description, methods and compositions are disclosed that for the first time allow successful in vitro cultivation of hemocytoblasts, leading to their proliferation and differentiation in progenitor cells and more mature hemocytes.

 At the end of the 1970s, cell suspension cultures were obtained for growing in vitro hematopoietic bone marrow cells. These cultures are very effective both in studies of the hematopoiesis process of healthy and leukemic cells, and in experiments on bone marrow transplantation, for example, for retroviral gene transfer. These cultures made it possible to study hematopoiesis in mice in detail and fully understand their hematopoiesis system. Moreover, the studies performed allowed for the retroviral gene transfer to cultured mouse bone marrow cells. As a result of this, it became possible to obtain labeled murine hematopoietic cells that support the existence of a multinucleated stem cell and to study various genes during leukemogenesis.

 And although it became possible to carry out retroviral genes into cultured murine osteocytes, such transfer was still possible in human bone marrow cells, since until now dexter-type cultures (long-living bone marrow cell cultures) are limited both by their lifespan and their ability to provide stem cell survival and the production of progenitor cells over the entire life span.

 Suspension cultures of human bone marrow cells, as it was established at the initial stage, have a limited hematopoietic potential, as a result of which the yield of progenitor cells and mature hemocytes decreases, and cell synthesis ceases by the 6-8th week. Subsequent modifications of the initially obtained suspension culture led only to improved cultivation methods. The solution to this problem is of such great importance that it is impossible to evaluate, since it would allow, for example, to replicate human stem cells and progenitor cells for bone marrow transplantation and protection against chemotherapy, to choose and conduct various manipulations with such cells, that is, to use gene transfer and synthesize mature human hemocytes for transfusion therapy.

 As a result of studies of hematopoiesis and cultivation of bone marrow cells, it was found that within the adhesive layers, fibroblasts and endocytes act as central elements of the cell stroma. These cells both create active binding sites for the growth of hematopoietic cells, and they can be induced to secrete hematopoietic growth factors that stimulate the proliferation and differentiation of progenitor cells. These hematopoietic growth factors include granulocyte colony stimulating factor (G-CSF), granulocyte macrophar colony stimulating factor (GM-CSF) and interleukin-6 (IL-6).

 The cultivation of human osteocytes on such adhesive layers in vitro, however, did not give encouraging results. In contrast to similar cultures of related species, such as shrews and blunts, suspension cultures of human osteocytes are not capable of producing a significant amount of both hematopoietic progenitor cells and clonogenic progenitor cells not producing a substrate for 6-8 weeks. And although from the literature it is known about continuously producing cultures for 3-5 months, culture media that provide stable continuous synthesis of progenitor cells from stem cells for more than 4-6 weeks are not reported anywhere.

 Moreover, the extracellular synthesis and production of progenitor cells during even such a short life span of these cultures is usually reduced so much that it is understood that these cultures generally do not provide stem cell survival or proliferation. In addition, non-activated bone marrow stromal cells studied in an isolated state secrete a small amount, if any, of hematopoietic growth factors (HGF).

 The lack of stable synthesis of precursor cells and mature blood cells in these cultures gave convincing evidence that they were not able to provide support for the continuous regeneration and increase of stem cell production. Based on this, it was suggested that these cultures either do not contain a critical stimulator (s) of stem cells and / or contain a new inhibitor (s) of them. But along with the explanation of the failure to identify cultures of hematopoietic growth factors and non-inducible stromal cells, a null hypothesis was put forward that combined the failure to detect HGF and the relative drawbacks of suspension cultures of human osteocytes that such continuous cultivation systems used in vitro do not fully restore hematopoiesis of the original cells of the bone marrow stroma in vivo.

 An increase in stem and progenitor cells for bone marrow transplantation is a potential area for the use of long-lived cultures of human osteocytes. Currently, autologous and allogeneic transplantation of human bone marrow is used as a treatment for diseases such as leukemia, lymphoma, and other life-threatening systemic diseases. However, such therapy requires the presence of a large number of donor bone marrow, which would provide the required number of cells for their successful engraftment.

 A culture that provides an increase in the production of stem cells and progenitor cells reduces the need to take a large number of donor bone marrow and allows you to take a small number of bone marrow cells, and then increase the number of stem cells and progenitor cells in vitro before administration to their recipient. It is also known that a small number of stem cells and progenitor cells circulate in the circulatory system. If such cells could be collected by electrophoretic methods and their volume increased, then it would be possible to obtain the necessary number of stem cells and progenitor cells for transplantation from peripheral blood, and there would be no need to take a donor bone marrow.

 When bone marrow transplantation is necessary, the introduction of approximately 1x10 - 2x10 mononuclear bone marrow cells per 1 kg of patient weight for their engraftment. To obtain such a number of cells, it is necessary to take a donor bone marrow in an amount of about 70 ml of brain per 70 kg of donor weight. And although 70 ml is a small fraction of the bone marrow of donors, the procedure for taking it requires a significant investment of time and replenishment of large blood loss in the process of its implementation. If the production of stem cells and progenitor cells could be increased by 10 times, then this procedure would be significantly reduced and, possibly, would be reduced only to the collection of such cells from peripheral blood, followed by their propagation in vitro.

 An increase in the production of progenitor cells is also suitable as a replacement maintenance therapy for chemotherapy and represents another aspect of the application of long-lived human bone marrow cell cultures. The dilemma faced by oncologists is that most chemotherapeutic drugs used to destroy cancer cells have a detrimental effect on all cells passing through the cell division cycle. The bone marrow is one of the most productive tissues in the body and, therefore, often refers to the body, which is primarily destroyed by chemotherapeutic drugs. This leads to a rapid disruption of the hematopoiesis during treatment with chemotherapeutic drugs, and therefore it is necessary to interrupt the course to restore hematopoiesis before continuing such treatment. It may take a month or more so that resting stem cells can raise the white blood cell count to the required level in order to resume chemotherapy, during which the number of red blood cells decreases several times. Unfortunately, although white blood cells are restored only between courses of chemotherapy, cancer cells have time for their growth, and it is possible to increase their resistance to chemotherapy due to their natural selective ability.

 To shorten the intervals between chemotherapy courses, a large number of progenitor cells and immature blood cells could normalize the patient's condition. This would have an effect on a significant reduction in the time intervals when the patient has a low rate of blood cells, which would allow him to resume chemotherapy faster. The longer the chemotherapy course and the shorter the intervals between chemotherapy doses, the greater the likelihood of a successful cure for cancer. Hematopoietic cells needed to increase the production of progenitor cells can be obtained either by taking donor bone marrow, or by collecting cells from peripheral blood. Bone cell harvesters harvest approximately 4x10 granulocyte macrophage colony forming units (CFU-GM) precursor cells. With electrophoresis of 5 L of peripheral blood, approximately 10 CFG-GM is usually collected, although this amount can be increased to 10 by prior administration to the donor of the GM-CSF factor. In order to quickly normalize the patient's condition, an injection of about 1x10 - 5x10 CFU-GM is necessary, which is about 100-1000 times more than the amount obtained by the routine procedure of taking bone marrow from a donor or collecting cells from peripheral blood. Thus, an increase in cell production from donor bone marrow or their collection from peripheral blood to increase the amount of CFU-GM by 2-3 orders of magnitude has a significant impact on the use and treatment of cancer with chemotherapy drugs.

 Gene therapy belongs to the fastest growing field of medicine, and the therapeutic potential of which is also difficult to assess. Gene therapies have many potential uses in the treatment of diseases (see, for example, Boggs, Int. J. All Cloning (1990), 8: 80-96, Kohn et al., Cancer Invest. (1989), 7 (2) : 179-192, Lehn. Bone Marrow Transp. (1990), 5: 287-293 and Verma, Scientific Amer., (1990), pages 68-34. Genetically transformed human stem cells have a wide range of potential applications in clinical medicine as gene therapy drugs.

 However, there are several problems that must be overcome in order to successfully conduct gene therapy. The first problem is the ability to introduce the desired treatment gene into the target cells. Secondly, the indicated gene must be adequately expressed in the target cell, as a result of which the required concentration of the gene product is ensured. And finally, the produced RNA or protein must be properly processed by the target cell to ensure their functional activity, that is, for gene therapy to influence the clinical picture of the treatment of the disease. Table 1 shows several methods for inserting a gene into human cells in vitro.

 Also, many such methods, for example, homologous recombination, are under development in many laboratories. Research in the field of gene therapy has been carried out for several years on several types of cells in vitro, then continued on animals, and, finally, recently entered the first stage of clinical testing in humans.

 The hematopoietic system is an ideal material as a gene product delivery system in gene therapy. Hematopoietic cells are readily available by drawing bone marrow or by collecting mononuclear cells from peripheral blood. Immediately after the genetic insertion in vitro, the treated cells can be reintroduced by intravenous infusion, after which the genetically transformed cells return to their place and continue to grow in the bone marrow. As mature blood cells circulate throughout the body, genetic modified cells can deliver a specific gene product to any target tissue.

 Most importantly, hematopoietic tissue contains stem cells that have great potential (possibly unlimited) for self-healing. By this we mean that if stable transduction of genetic material into such stem cells is ensured, then with the reinfusion of hematopoietic tissue these modified stem cells are capable of proliferation and repopulation in the brain tissue together with cells that provide expression of the new gene. This leads to the long-term, possibly throughout their life cycle, delivery of the desired gene product. Similarly, successful stable gene transfer to stem cells located in other organs, or embryonic stem cells, also leads to continuous, long-term delivery of the gene product.

 Gene therapy using hematopoietic stem cells has a wide range of applications both for the treatment of diseases specific to the hematopoietic system, as well as systemic diseases of other organs. Within the framework of the hematopoietic system, gene therapy with stem cells can be used to treat both hereditary and acquired diseases. For example, conditions associated with hemoglobin deficiency, in particular alpha and beta thalassemia, can be treated by introducing a gene encoding the alpha or beta chain globin in combination with regulatory sequences that transmit high tissue-specific affinity to red blood cells (see Grosvell et al., Cell, (1987), 51: 975-986). Similarly, sickle cell anemia can be corrected by genetic insertion of the fetal globin gene into hematopoietic stem cells, since the regulated expression of high concentrations of embryonic hemoglobin is sufficient to prevent the production of sickle cell erythrocytes despite the presentation of sickle cell hemoglobin, etc. Molec. Biol. (1979), 133: 435).

 Genetic impairment of neurophiles caused by insufficient functional activity of proteins, such as deficiency of the lymphocytotoxic determinant (LAD) or chronic granulomatosis (CGD), can be successfully treated by genetic insertion of a gene encoding a defective or lost gene, in combination with regulatory DNA sequences that transmit a high degree tissue-specific expression into hematopoietic stem cells (see Wilson et al., Science (1990), 248: 1413-1416). Inherited diseases, including thrombopathy, such as angiohemophilia, can be treated by insertion of a gene encoding, for example, Billlebrand factor in combination with sequences that regulate its expression and secretion.

 The suitability of hematopoietic stem cell-based gene therapy to replace congenitally defective genes in the treatment of lymphocyte-mediated immunodeficiency complexes, for example, as pronounced immunodeficiency states due to a lack of adenosine deaminase, is especially obvious. Retroviral gene therapy of circulating T cells in combination with a gene encoding adenosine deaminase has been found to be effective in reducing the clinical picture in patients with immune deficiency, however, these effects are only temporary, since transfected T lymphocytes have a limited in vivo life (see Kasid et al., Proc. Nat. Acad. Sci. (USA) (1990), 37: 473-477, or Culver et al., Proc. Nat. Acad. Sci. (USA) (1991), 38: 3155-3159). If the indicated gene could be successfully transferred to such hematopoietic stem cells, then any T-cell produced from these stem cells would contain and express the adenosine deaminase gene. Thus, due to the fact that transfected stem cells have the ability to survive and rapidly multiply throughout the patient's life, T-cell-mediated ADA deficiency is usually treated with maintenance therapy by transferring the gene to the stem cell (see Wilson et al., Proc . Natl. Acad. Sci., (USA) (1990), 87: 439-443).

 In addition to congenital disorders of the enzymatic processes of the hematopoietic system, the stem cell-based gene therapy method could be used to protect embryonic cells and their progenitor cells from the toxic effects of exogenous factors, such as viruses and chemotherapy. For example, it has been found that gene transfer of the DNA sequence encoding the antigen-reactive T-lymphocyte (TAR) binding site of the HIV transactivating factor TAT provides protection of T cells from spreading HIV infection (see Sullenger et al., Cell, (1990 ), 63: 601-608). Stable transduction of these sequences into hematopoietic stem cells leads to the aggregation of T cells produced from these stem cells, which are relatively or completely resistant to the spread of HIV infection.

 Similarly, the successful transfection of genes encoding the combination drug resistance gene (MDR) into hematopoietic bone marrow stem cells that are relatively resistant to the effects of anti-cancer chemotherapeutic treatment. After autologous bone marrow transplantation based on such genetically transformed cells, patients will be able to more easily tolerate chemotherapy drugs, from which stem cells were protected due to the deep inhibition of bone marrow function caused by these anticancer drugs. As a result of this, patients will be able to take more effective dosages of anticancer chemotherapy drugs with less toxicity.

 Everyone can easily understand that gene therapy based on hematopoietic stem cells is also suitable for the treatment of acquired diseases of the hematopoietic system, for example, leukemia, lymphoma and aplastic anemia. Immediately after establishing the genetic nature of these diseases, the introduction of a gene product that either eliminates the cause of the pathology of the gene in the cell, or the gene itself (possibly by splicing or replacing the gene itself), provides the correction of the deviation from the norm.

 At a broader level, however, hematopoietic stem cell-based gene therapy can also be used to treat non-hematopoietic diseases. Gene transfer of DNA sequences carrying drug soluble proteins can result in mature blood cells that continuously secrete the required number of treatment molecules. So, for example, this method can also be used to treat, for example, diabetes mellitus by introducing insulin DNA sequences in combination with regulatory DNA sequences that ensure proper expression of the transfected insulin gene, possibly in response to an increase in blood sugar. Systemic hypertension can be treated by genetic insertion of stem cells with DNA sequences encoding secretory peptides that act as a competitive inhibitor with respect to angiotensin convertase, calcium tubules of the smooth muscle vasculature, or adrenoreceptors. In all likelihood, Alzheimer's disease can be treated by genetic insertion into stem cells of DNA sequences encoding enzymes that destroy amyloid plaques in the central nervous system.

 Thus, there are many applications of gene therapy, in particular the introduction of a gene product into stem cells (see above Boggs and others, Kohn and others, and / or Verma and others). And, of course, there are many cases of some progress in ensuring the transfer of the treatment gene into differentiated human stem cells, as described, for example, in the case of T-lymphocytes (see, Kalsd et al., Proc. Nat. Acad. Sci. (USA) (1990), 37: 473-477, and Culver et al., Proc. Nar. Acad. Sci. (USA) (1991), 88: 3155-3159).

 Unfortunately, prior to the creation of the present invention, the provision of stable gene transfer to human stem cells was unsuccessful. Although several research groups have demonstrated the feasibility of implementing retrovirus-mediated gene transfer into human hematopoietic cells, an attempt to transfect primitive elements of human hematopoietic stem cells has failed. The results of such studies are very different from experiments conducted in mice, which showed the possibility of retrovirus-mediated gene transfer into human hematopoietic stem cells (see Wilson et al., Pro. Nat. Acad. Sci. (USA) (1990), 87 : 439-443).

 The main difficulty for the successful implementation of gene therapy based on human hematopoietic stem cells was the inability to ensure the insertion of genes into human hemocytes under conditions of division and proliferation of stem cells. For successful sustained insertion of a gene into a target cell, it is necessary that such a target cell undergo at least one cycle of cell division. Thus, if the stem cells do not divide in the presence of the target genetic product, then this product is introduced unstably into such stem cells. Before the creation of the present invention, there was no system that would support ex vivo division and proliferation of human stem cells and would allow for the successful genetic transformation of such stem cells.

 The literature relevant to the present specification also includes US Pat. No. 4,721,096, which discloses a 3-dimensional system comprising stromal cells for the growth of hemocytes. See also sources contrasted in this description, Glanville et al., Nature (1981), 292: 267-269 - describes a gene product of murine metallothionein; Wong et al., Science (1985), 228: 810-815 describes the human factor GM-CSF; Lemischka et al., Cell (1986), 45: 817-927 describes retrovirus-mediated gene transfer as a marker of hematopoietic stem cells and a technique for monitoring the development of these cells after transplantation; Yang et al., Cell (1986), 47: 3-10 describes the preparation of human interleukin-3; Chen et al., Mol. Cell. Biol. (1987). 7: 2745-2752 describes the transformation of mammalian cells with plasmid DNA; Greaves et al., Cell (1989), 58: 979-986 describes the human CD2 gene; Civin et al., J. Immunol. (1984), 133: 1576 discloses the CD34 antigen; Martin et al., Cell (1990), 63: 20-211 describes human S-CSF; Forrester et al., J. Cell Science (1984), 70: 93-110 describes a flow chamber for parallel immunoelectrophoresis; Coulembel et al., J. Clin. Invest., (1986), 75: 961 describes a deficiency of CML cells in static cultures.

 In view of the foregoing, there is a great need for the development of methods and compositions for ex vivo replication and stable genetic transformation of human stem cells and optimization of cultures of human hematopoietic progenitor cells, especially in light of the huge potential for increasing the yield of stem and progenitor cells, as well as expanding the capabilities of gene therapy that these systems provide. Unfortunately, to date, attempts to solve these problems have not met expectations.

Disclosure of the invention
In accordance with the foregoing objective of the present invention is the creation of new methods, including culture conditions and reactors, for ex vivo replication and stable genetic transformation of human stem cells.

 Another objective of the present invention is the creation of new methods, including culture conditions and reactors, to optimize (I) cultures of human hematopoietic progenitor cells and (II) stably genetically transformed human hematopoietic progenitor cells.

 The present invention is based on the results of studies conducted by the author, during which new methods were developed, including culture conditions and reactors, for ex vivo replication and stable genetic transformation of human stem cells and / or for optimizing cultures of human hematopoietic progenitor cells. These methods are based on the cultivation of human stem cells and / or human hematopoietic progenitor cells in a liquid culture medium, which is replaced, preferably perfused, either continuously or periodically at a flow rate of 1 ml of medium per 1 ml of culture after about 24 to 48 hours subsequent removal of metabolites and replenishment of nutrient deficiencies, while maintaining a physiologically acceptable state of the specified culture. In a particularly preferred embodiment of the present invention, hematopoietic growth factors are added to said culture medium while replacing the above medium.

 During the creation of the present invention, the authors unexpectedly found that an increase in the metabolic rate in the specified environment, which is used in accordance with the present invention, with the optional addition of hematopoietic growth factors in a rapidly changing culture medium (1) ensures the viability of the cultures in which human stem cells grow over a long period of time - about 5 months, (2) provides support for the cultures in which human generation occurs hematopoietic progenitor cells by dividing and differentiating human stem cells over a long period of time of about 5 months and (3) stimulates an increased metabolism of human stromal cells and the release of GM-CSF factor from them, including from stroma cells of the human bone marrow . The present invention allows for the first time to ensure the survival of human stem cells and their proliferation in culture.

 The present invention also provides an ex vivo system that provides continuous proliferation of human stem cells, which allows the successful introduction of a genetic product into human stem cells, thereby producing stably genetically transformed human stem cells. This embodiment of the present invention can be used to transfer any genetic material that can be introduced either by genetic engineering into a recombinant retrovirus or any gene transfer vector that requires cell division. Genetically transformed human stem cells obtained in accordance with the proposed method can be used to treat a wide range of the above diseases.

 The present invention also provides methods for increasing the efficiency of transferring genetic material into human hematopoietic progenitor cells in combination with methods that ensure stable genetic transformation of human stem cells and / or human hematopoietic progenitor cells.

 The present invention also provides bioreactors and compositions that provide for the efficient proliferation of human cells in culture, especially cells in the early stages of their maturation, including stem cells. These methods use stromal cells transformed by standard techniques that provide constant or inducible production of growth factors, and these cells are isolated to allow easier isolation of hematopoietic cells. Providing continuous perfusion and recycling of the corresponding cells, it is possible to significantly increase the density distribution of viable hematopoietic cells and their output. In the bioreactor, a protein carrier for stromal cells is used as a starting material. This protein membrane or other substrates provide the separation of stromal and hematopoietic cells.

 A brief description of the drawings.

 In FIG. 1 is a schematic view of a perfusion chamber.

 In FIG. 2 shows a schematic view and a flow chart of the passage of perfusion solution.

 In FIG. 3A shows a schematic view of a flow chamber for measuring shear stress during cell separation.

 In FIG. 3B is a side view of the flow chamber of FIG. 3A.

 In FIG. 3C shows a graphic shear stress profile of hematopoietic cells.

 In FIG. 4A and 4B show horizontal and side projections of a flow chamber for growing and separating hematopoietic cells.

 In FIG. 5A and 5B show a perspective view of a flow chamber where substrates are sequentially removed to allow continuous growth of stromal cells.

 A preferred embodiment of the invention.

 The advantages of the present invention can be observed when used in relation to any standard liquid culture system of human stem and / or progenitor cells, for example, as established in hematopoietic cell culture (s). Using a culture-exchange quick-change technique that is used according to the invention in combination with optionally adding hematopoietic cell growth factors to it, the authors unexpectedly discovered that this allows the creation of standard suspension culture systems for human hematopoietic cells that include cultures that operate in the presence or absence of animal serum or plasma, including equine, bovine, fetal calf, or human serum.

Suspension cultures of human hematopoietic cells that are suitable for use in accordance with the present invention can be prepared at a cell distribution density of 10 ⁴ to 10 ⁹ cells per ml of culture using standard well-known components to obtain culture media, for example, Dulbecco's medium modified by the Isca method (IMDM), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640 Medium, Alpha Medium or McCoy's Medium, which can be used in combination with serum albumin, cholesterol and / or lecithin, selenium and inorganic salts. As indicated previously, corticosteroids, for example, hydrocortisone at a concentration of 10 ^-4 to 10 ^-7 M, or other corticosteroids in the same dosage, for example cortisone, dexamethasone or solymedrol, can be added to these cultures. Cultivation is usually carried out at a pH approaching the pH of the body, that is, in the range from 6.9 to 7.4. The culture medium is usually maintained in an oxygen-containing atmosphere, comprising from 4 to 20 vol.%, Preferably from 6 to 8 vol.% Oxygen.

Using these standard growing methods, the used cell mass can be replenished to the required size, for example, up to 10 ³ times or even more, either by stem cell content or hematopoietic progenitor cells. To ensure such enrichment, well-known methods can be used in accordance with either the methods of selective negative reaction or selective positive reaction. So, for example, for a selective negative reaction, mature cells are removed using immunoassay methods, for example, by labeling in stem and progenitor pools without using a microplate with mouse anti-human monoclonal antibodies, followed by removal of mouse coated cells with antibody when they adhere to plastic plates coated with rabbit anti-mouse Ig (see, for example, Amerson et al., J. Clin. Invest. (1985), 76: 1286-1230).

 The basis of the invention is a fundamental change in the conditions for the cultivation of suspended human bone marrow cells using any of the above conditions for maintaining the culture, rapid replenishment of the nutrient medium. In accordance with the standard cultivation method of the present invention, it is necessary to replace the nutrient medium and serum every week, that is, either carry out their complete replacement weekly, or half the medium and half serum twice a week. In accordance with the invention, the culture medium of said culture is preferably replaced in the form of a perfusate, either continuously or periodically, at a flow rate of about 1 ml of medium per 1 ml of culture for about 24 to 48 hours, and the density of cultured cells is from 2x10 to 1x10 cells per 1 ml of medium. To ensure a cell distribution density of about 1x10 to 2x10 cells per 1 ml, the same amount of culture medium can be used when replenished. To increase the cell density of more than 10 cells per ml, it is necessary to increase the flow rate of the medium being replaced proportionally, in order to ensure the flow rate of the medium and serum at a constant level per 1 cell. per unit of time.

 The replenishment of the nutrient medium in accordance with the invention can be carried out by any method that ensures its restoration, for example, by taking an aliquot of the spent culture medium and replacing it with a fresh aliquot. The indicated aliquot can be added by gravity either by means of a pump or other suitable means. The flow of the medium can flow in any direction, or you can use many streams supplied from different directions depending on the configuration and media culture. Preferably, the fresh nutrient medium is supplied in such a way that its contact with the whole cell mass is ensured. In the most preferred embodiment, the nutrient medium is added to the culture like a blood circulation process that takes place in vivo, that is, it is perfused through at least part of the cell mass, and it is preferable that it passes through the entire biomass.

 Another optional but important aspect of the implementation of the present invention is that hematopoietic growth factors, including synthetic growth factors, are added to cultures with a fast metabolic process. In a particularly preferred embodiment of the present invention, IL-3 and GM-CSF cytokines are added to such a culture along with the culture medium at a flow rate of 0.1 to 100 ng / ml / day, preferably 0.5 to 10 ng / ml / day, and most preferably from 1 to 2 ng / ml / day. Erythropoietin (Epo) can be added to the nutrient medium in an amount of from 0.001 to 10 units / ml / day, preferably from 0.5 to 0.15 units / ml / day. Mast cell growth factors (MCF, c-complement ligand, Still factor) can be added to the culture medium in an amount of 1 to 100 ng / ml / day, preferably 10 to 50 ng / ml / day. IL-1 (a- or b-) can also be added to the nutrient medium in an amount of 100 u / ml over 3-5 days. In addition, fibroblast growth factors, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF or EGF in an amount of 1 to 100 ng can be added to the culture medium. / ml / day.

 During the development of the present invention, it was found that when IL-3, GM-CSF and Epo are used in accordance with the conditions described above, differentiation-specific growth of red blood cells can be achieved. Alternatively, if IL-3 and GM-CSF are used in the presence or absence of IL-6 or G-CSF, said culture produces predominantly granulocytes. It was also established that the cultures of the invention throughout the whole time lost T- and B-lymphocytes.

 The level of metabolic products in the specified environment is usually kept within a given range. The glucose content is usually kept in the range from about 5 to 20 mm. The concentration of lactate is usually kept at less than 35 mm. Glutamine levels are usually maintained in the range of about 1-3 mM. The concentration of ammonia is usually kept at less than 35 mm. These concentrations can be controlled by either periodic measurements or continuous real-time measurements of the content using conventional methods (see, for example, Caldwell et al., J. Cell, Physiol. (1991), 147: 344-353).

 As cells that can be grown according to the present invention, you can use any human stem cells, or cell mass containing such stem cells, including human peripheral blood mononuclear cells, human osteocytes, human liver embryonic cells, embryonic stem cells and / or human sickle cells. Each of these cell compositions contains stem cells and / or human hematopoietic progenitor cells. In other cellular compositions containing human stem cells, any human human bone marrow stem cell can also be used in accordance with the invention.

 In a preferred embodiment of the invention, said cell culture can be enriched by additional stem cell production in said cell composition. Such culture enrichment can be provided in accordance with the methods described above, and such an enriched culture, if used in accordance with the present invention, is primarily suitable as a means for gene therapy by transferring gene material into human stem cells, including stem cells contained in human bone marrow and in human bone marrow stem cells. As stem cells of the human bone marrow, cells can be used that can be isolated from human bone marrow tissue, from peripheral blood, embryonic hepatocytes, or the blood corpuscle.

 Generally speaking, in this aspect of the present invention, to ensure sustainable genetic transformation of human stem cells to such cells cultured in accordance with the proposed methods, a retroviral infected cell line or supernatant obtained by culturing such a malformed cell line is added , or any other gene transfer vector. The present invention allows to increase the content of stem cells and the replication of human hematopoietic progenitor cells, however, in contrast, known cultures provided replication of human hematopoietic progenitor cells with a decrease in its intensity (that is, "decaying cultures"). The proposed cell culture technique allows for the first time to provide an increase in cell mass in a culture requiring retrovirus infection of the cells. Until now known systems where retrovirus infection was performed on decaying cultures did not provide infection of cells in the early stages of their growth. The present invention, especially if it is implemented in combination with the enrichment of a stem cell pool, and even more so when hematopoietic growth factors, including synthesized growth factors, are added, this makes it possible to obtain a very effective agent for stem cell infection in vitro.

 During the development of the present invention, it was found that the addition of supernatants containing recombinant retroviruses to these cultures lead to the introduction of viruses and the genes that they carry into human (hematopoietically) stem cells. Precursor cells formed during division and differentiation from these stem cells, and mature blood cells formed from subsequent division and differentiation of these predecessor cells, contain transfected DNA sequences throughout the cultivation of hematopoietic cells in vitro. It was also established that when a retrovirus is administered only at the initial stage of cultivation, transfected progenitor cells and mature blood cells that can leave stem cells are obtained, proliferation and stable genetic transformation of which was ensured only at the beginning of biosynthesis, since there was no infection with a retroviral pathway a cell cannot infect a neighboring cell. Thus, precursor cells and more mature cells carrying the target genetic material took the gene from more primitive stem cells that were genetically transformed at the initial stage of retroviral infection.

 In a preferred embodiment of the invention, human hematopoietic cells, either isolated from bone marrow tissue, peripheral blood, liver embryonic cells or umbilical cord blood, are first enriched with respect to increasing stem cell content by removing more mature hemocytes. Such enrichment is carried out by incubating hematopoietic cells with murine monoclonal antibodies that recognize epitopes on mature nemocytes and bone marrow progenitor cells, rather than stem cells. After this, labeled cells are removed by immunoadhesion with a rabbit anti-mouse Ig immunosorbent surface. The resulting cells with a negative reaction to differentiation (Lin-) are then incubated in the presence of a retroviral or other gene transfer vector in accordance with the invention. In a preferred embodiment, the cultivation is performed in the presence of GM-CSF, preferably in an amount of 1 mg / ml / day) and IL-3 (preferably in an amount of 1 mg / ml / day) with or without IL-1 (preferably in a dose of 50 units / ml over a 4-day period), as well as in the presence or absence of a c-complement-binding ligand (mast cell growth factor) (preferably at a dose of 10 units / ml / day).

 Retroviral infection can be carried out either by introducing supernatants into the indicated culture medium (for example, from 5 to 20%, v / v) obtained from a cell line with a packaging defect infected with recombinant retrovirus within 2-21, preferably 10-14 days from the start culturing either by growing Lin cells directly on this same previously indicated cell line, or using both.

 The use of retroviral supernatants is preferred and the incubation period in the presence of the virus is 12 to 16 days. The incubation of cell lines with packaging defects is preferably carried out under conditions close to the state of confluence, with the replacement of the nutrient medium, after which the incubation is carried out for another 12-15 hours. After this, the culture medium is collected and used for transfection of human stem cells. In accordance with the invention, any (well-known retroviral cell lines with packaging defects) can be used, and their cultivation can be carried out by any known method (see, for example, Wilson et al., Science (1990), 248: 1413-1416 and / or Sullenger and et al. Cell (1990), 63: 601-608. As an example of such packaging defective cell lines, NIH 3T3 cell lines and kidney carcinoma cell line 5337 can be mentioned.

Any gene that is introduced into a recombinant retrovirus together with appropriate promoters and enhancers that ensure its expression can be introduced into human stem cells and hematopoietic progenitor cells. The present invention for the first time proposes conditions that ensure the viability of stem cells and their proliferation
in these cultures, which makes it possible to obtain stably transfected and genetically modified stem cells in such cultures. The terms “stably transfected” and “stably transformed” are used in this context to emphasize that the introduction of exogenous DNA into the chromosomes of human stem cells has been made possible by the present invention because it allows exposure of this DNA to dividing human stem cells ex vivo.

 In accordance with the invention, it is possible to obtain cultures in which the production of human hematopoietic progenitor cells occurs by dividing and differentiating them from stem cells over the entire cultured period of time, and for at least 5 months.

 The data obtained indicate that the perfusion rate of the medium varies very significantly in determining the behavior of extracellular cultures of human bone marrow. These data showed that when the rate of renewal of the indicated medium increased from the traditional once a week dexter type to the frequency of medium replacement daily on the order of 7 volumes per week, a significant positive effect of the hematopoiesis ex vivo process was obtained. In experiments conducted during the development of the invention, all cultures showed significant cell loss during the first 3-4 weeks. After this decline, cultures stabilized, and the effect of perfusion intensity in the medium became more pronounced. The frequency of updating the growth medium of 3.5 volumes per week led to the greatest proliferation of cell cultures, and, in addition, to the longest continuous period of cultivation in terms of the production of progenitor cells. It should be noted that during the time interval from 4 to 10 weeks the number of non-adherent cells produced twice a week was practically stable or tended to increase.

 During the entire cultivation period, the total number of cells produced after 3.5 weeks almost tripled in comparison with the traditional dexter-type cultivation technique. Moreover, a stable yield of progenitor cells lasts up to 18 weeks.

Human stromal cells, for example, which are identified in the tissue of the human bone marrow, may or may not necessarily be included in the cultures of the invention. In standard cultures, such cells are contained in an amount of about 10 ⁴ to 10 ⁹ (stromal cells of the total cell content).

 In another aspect of the present invention, the proposed cell cultures are surprisingly found to lead to an increase in metabolic rate and secretion of GM-CSF and IL-6 from human bone marrow stromal cells. While the presence of GM-CSF factor in the supernatant of stromal cells of the human bone marrow was not detected, the rapid updating of the nutrient medium in accordance with the invention leads to the stimulation of stromal cells of the human brain with a release of 300 ng / ml / day to 200 ng / ml / day factor GM-CSF. The secretion of IL-6 by stromal cells of the human bone marrow also increases with rapid updating of the nutrient medium in accordance with the invention from 1-2 ng / ml / day to 2-4 ng / ml / day. Such an increase is noted both in the case when only a quick update of the nutrient medium is carried out, and in the case when the nutrient medium is rapidly updated with the simultaneous introduction of hematopoietic growth factors. Based on the data obtained by the authors of this invention, the effect of rapid replacement of the nutrient medium on the production of cytokines from human stromal cells can be observed using such stromal cells in any complex tissue culture system.

 As an example, the nutrient medium used in the present invention may contain three main components. The first component includes Dulbecco's Medium, Claims Modified (IMDM), Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640 Medium, Alpha or McCoy's Medium, or an equivalent component for preparing known culture media. The second component is the serum component, which includes at least horse or human blood serum, and if desired, it may also include fetal calf serum, newborn calf serum and / or bovine serum. As the third component, you can use corticosteroids, for example hydrocortisone, cortisone, dexamethasone or solymedrol, or combinations thereof, but the use of hydrocortisone is preferred.

 The composition for the preparation of various nutrient media that can be used is shown in table A.

 The serum component may be present in the culture in an amount of at least 1 to 50% (v / v). The serum concentration in the culture may preferably be from about 15 to 30% (v / v). With an increase in the concentration of the serum component, the metabolic rate increases proportionally. The third component of the culture may be present in an amount of from 10 to 10 M, and preferably from 5x10 to 4x10 M. This component of the culture medium is used up to balance, as a result of which all three of these components add up to 100%. Alternatively, the whey component can be replaced with any of several standard whey replacement mixtures, which usually include insulin, albumin and lecithin or cholesterol (see, for example, Migliaceio et al., Exp. Hematol. (1990), 18: 1043 -1045, Iscove et al., Exp. Cell Res. (1980), 126: 121-126 and Dainiak et al., J. Clin. Invest. (1985) 76: 1237-1242).

 As an illustration, the concentration of human hematopoietic stem cells can be increased as follows. Red blood cells are isolated from bone marrow puncture by centrifugation in a density gradient of ficol-hypak, after which mononuclear cells are incubated with a cocktail of antibodies that recognize mature hemocytes, including red blood cells, granulocytes, macrophages and mature lymphocytes (both B- and T- cells). In addition, antibodies that recognize concurrent progenitor cells (including anti-CD33) are included within the scope of the present invention. Mature cells are then removed by any of a variety of well-known methods, including panning, magnetic bead immunobinding, or cell sorting using a laser fluorescence intensity sorter (FACS). With the removal of mature blood elements, the possibility of infection with a recombinant virus is significantly increased, and, therefore, the transfer of the desired gene (s).

 In another embodiment, the present invention provides methods for growing hematopoietic cells in culture using fibroblastocytes, usually modified, that allow the incorporation of growth factors together with protein components into said mixtures of fibroblast and hematopoietic cells and practically continuous perfusion, with recycling if desired, and thereby provide a stable effective state of the growth medium.

 The description of the proposed method of the present invention can be divided into several parts, which relate to the conditions for perfusion, the bioreactor and its construction of the transformation of fibroblasts.

 The inventive bioreactor includes a reaction vessel, which can be of any design that allows for the necessary distribution of cells, the introduction of nutrients and oxygen, the removal of waste metabolic products, the optional recycling of hematopoietic cells, the replacement of stromal cells and the collection of hematopoietic cells. Such a reactor should provide conditions that are almost close to bone perfusion. In vivo, approximately 0.08 ml of blood serum is consumed per 1 ml of bone marrow per minute, or, to put it differently, 0.3 ml of serum per 10 cells daily. Such a medium, therefore, should be replaced on average by at least 50%, preferably 100%, every 24 hours to stabilize the normal level of metabolic products, which should not be limited in growth. Replacement flow rates typically range from 0.5 to 1.0 perfusate per 10 cells daily, which is similar to empirical in vivo perfusion calculations.

The perfusion rate in the bioreactor varies depending on the density of cells in the reactor. For cells grown at a flow rate of 2-10 · 10 cells / ml, this indicator is 1 ml / ml of reactor volume (24-48 hours, if the medium used contains 20% serum, or 10% fetal calf serum and 10 % horse serum, or 20% only fetal calf serum). With increasing cell density, the perfusion rate also increases proportionally, which ensures a constant consumption of serum per cell per unit time. So, if cells are cultured at a density of 5 · 10 cells / ml of medium, the perfusion rate is 0.1 ml / ml of reactor volume per minute. The indicated flow rates are important for stimulating the endogenous production of hematopoietic growth factors from healthy stroma cells of the human bone marrow in culture. GM-CSF, as well as S-CSF, g-38-IL-6, G-CSF and other hematopoietic growth factors can be used as hematopoietic growth factors induced at such indicators of serum and medium consumption. Such norms are usually set in bioreactors so that the shear stress from the longitudinally flowing stream experienced by stem cells and progenitor cells at their attachment sites to the stromal cell is in the range of 1.0-5.0 dyne / cm ² .

 Many different media can be used to grow hematopoietic and stromal cells. Examples include MEM, IMDM and RPMI, which can be supplemented with combinations of 5-20% fetal calf serum, 5-20% calf serum and 0-15% horse and / or serum-free medium supplemented with PDGF, EGF , HGF, PGF and other growth factors to stimulate stromal and stem cells. In addition to the growth factors that transformed fibroblasts provide, additional growth factors can be used in the perfusion medium, especially in cases where "trained cells" require site-specific differentiation. The growth factors that can be included in such a perfusion medium either by their secretion by stem cells or by addition to it include GM-CSF, G-CSF or M-CSF, interleukins 1-7, especially 1, 3, 6 and 7, TGF-a or b, erythropoietin and the like, preferably human growth factors. Of particular interest is a combination containing about 0.5-2, preferably 1 ng / ml GM-CSF and 0.5-2, preferably 1 ng / ml, and also 0.1-2 u / ml / day of the final dilution of erythropoietin, from about 100-300 u / ml / day G-CSF and about 1-10 u / ml / day (a factor known as mast cell growth factor or kit-ligand). It is clear that one and several, preferably at least two growth factors will be maintained by secreting from transformed cells, which are contained in an amount sufficient to maintain the required level of these growth factors in this perfusion medium.

For convenience, the physiological temperature is maintained in the bioreactor, i.e. 37 ^° C, although lower temperatures can also be used, including 33 ^° C, but usually not lower than 25 ^° C. Humidity is usually 100% and the air contains approximately 5% carbon dioxide. The perfusion medium can be saturated with oxygen inside or outside the reactor, and various methods of internal oxygenation are provided. For example, oxygenation inside a bioreactor can be ensured by hollow fibers, porous sintered disks, through silicone tubes or other membranes with appropriate permeability and hydrophobicity. The level of nutrients and metabolists is usually maintained in a relatively wide range. The glucose content usually ranges from about 5 to 20 mm, usually from 10 to 20 mm, the concentration of lactate is usually kept below 35 mm, but its content can reach about 20%. The concentration of glutamine is usually maintained in the range from 1 to 3 mm, usually from 1.5 to 2.5 mm, while ammonium is approximately below 2.5 mm, preferably below 2.0 mm. The flow of the medium can come by gravity, using a pump or other methods, and it can move in any direction or in the form of many different flows coming from different directions depending on the packaging of the reactor. It is preferable to use a laminar flow, where the stream passes horizontally through the reactor or vertically from the bottom up of the reactor or vice versa.

When it is suspected that human hematopoietic cells contain neoplastic cells, leukolymphomas or carcinomas, the perfusion flow can be selected so as to isolate normal progenitor cells from neoplastic hematopoietic cells. Healthy hematopoietic progenitor cells have been found to attach to the stroma and matrix of proteins with an affinity that can withstand shear stress created by horizontal flow of about 1.5-2.0 dyne / cm ² In contrast, neoplastic cells and their precursors have significantly low affinity for stroma in the range of about 0.05-1.2 dyne / cm ² . By holding the perfusate feed rate, which provides an intermediate shear stress index between tolerated normal and neoplastic progenitor cells, typically greater than 1 dyn / cm ² , neoplastic progenitor cells can be separated from healthy progenitor cells by perfusion for at least about two days preferably for at least four days and more preferably for seven days or more. In this way, it is possible to increase the volume of healthy hematopoietic cells in a patient at the same time, using a suitable flow rate, to separate neoplastic cells.

To illustrate the use of shear stress to separate hematopoietic tumor cells from healthy hematopoietic cells, a case of chronic myeloid leukemia (CML) can be cited. Shift tolerance for CML cells varies from 0.05-1.2 dyne / cm ² . This difference allows you to effectively remove CML cells with an individual bone marrow sample. Using a shear rate of the order of 1.2-1.5, preferably 1.3 dynes / cm ² , the CML cell can be successfully separated.

 Resistance to shear stress in the cells of the bone marrow of an industrial patient can be determined using a radial cone-shaped flow chamber. In this chamber, the shear stress experienced by the cells decreases from a distance d from the beginning of the chamber as a function of 1 / d. The strips can then be analyzed for the size of the cell population, and the shear stress index determines at what level the given cell population should be.

 To separate leukemic stem cells, progenitor cells and stem cells from bone marrow puncture of patients with leukemia are placed in a radial flow chamber. This chamber consists of two parallel plates made of polycarbonate resin or glass.

Based on the results of these measurements, a cascade of parallel rectangular cells is established where the perfusate flow rate (see Figs. 4A and 4B) at a lower level creates a shear stress that is established in a cone-shaped chamber with the removal of leukemic cells from the stroma, but without affecting any of the healthy cells. In the case of the bone marrow of a patient with chronic myeloid leukemia, this shear stress is usually 1.01-0.05 dyne / cm ² . The actual perfusate flow rate depends on the size and geometry of the indicated cells. This patient’s bone marrow cells were grown in these rectangular cells at a concentration of 5x10 - 50x10 / ml in Dulbecco’s medium modified by the Iscov method with 5-20% (usually 10%) horse serum with or without 10 M hydrocortisone. These bone marrow cells were incubated within 12-24 hours without supplying perfusion solution, and after that perfusion solution was started. Cells were cultured for 3-7 days, removing all non-adhesive cells. Adhesive cells were removed from these rectangular plates by aspiration or mechanical shaking, after which they were collected. These cells can then be directly injected into the patient or stored in liquid nitrogen according to standard techniques before their direct use.

 In addition to the cells of the hematopoietic system, cells of other organs can also be separated by using the difference in tolerance to shear pressure. Thus, where there are distinct subpopulations of cells within a given complex of cells, the methods described above can be used to separate the types of test cells from a suspension culture obtained, for example, from skin tissue, liver, muscle, nerve tissue or epithelium. Of particular interest is the separation of cells within a population of healthy cells. A shared population of cells is brought into contact with the stromal substrate, as will be described later in the description, for example, as a purified protein or cellular component with which the test cells bind. The shear stress resistance of each of the adhesive subpopulations is determined by the method described above. The perfusate feed stream can then be adjusted to maintain the desired subpopulation of cells in the stroma. Target cells are then harvested as described above.

 Various packaging can be used in the proposed reactor to ensure adhesive cell growth, while providing some physical separation between stromal and hematopoietic cells, as well as for the possibility of some or close interaction between stromal and hematopoietic cells. In this case, factors released from stromal cells can be easily absorbed by hematopoietic cells to facilitate their proliferation and proper differentiation and maturation.

 The protein matrix used as a substrate for cells can take the form of collagen particles, for example sponges, and porous collagen beads, said sponges or microspheres consisting of extracellular protein matrix of bone marrow or protein-coated membranes, where collagen can be used as a protein, fibronectin, gemonectin, peptide, a mixture of protein with bone marrow matrix and the like. Membrane pore sizes typically range from 1 to 5 ms so that interactions between different types of cells can be achieved, although physical separation is still maintained.

 Protein-coated membranes can be used. Many materials can be used to produce membranes, such as polypropylene, polyethylene, polycarbonate and polysulfonate resins. The membranes must have sufficiently small pores through which the modified cells must not pass, however, they can grow and form a confluent layer on the side surface of such a membrane. Typically, the pores of such membranes range from about 1 to 5 ms. In this case, hematopoietic stem cells can grow on the opposite side of the membrane and interact with modified cells, as a result of which growth factors can be transferred directly from transformed cells to hematopoietic progenitor cells. Precursor cells, stem cells, have the ability to attach to lateral cytoplasmic projections that extend into these pores. Hematopoietic differentiation from stem cells occurs on one side of the membrane, and differentiated progenitor cells are not able to pass back through membranes, which are already mostly covered with a layer of stromal cells when the state of continuity is almost or already achieved. For example, you can set many cells in which stromal cells can grow, and hematopoietic cells can move in accordance with the cell that stromal cells have at a subconfluent level. Thus, in the presence of a moving membrane between these cells, when the stromal cells are approaching a confluent state, usually after about 8-12 weeks, it is possible to open or remove such a barrier between the cells and allow the stromal cell to move to a new cell, and the hematopoietic cells to come into interaction with subconfluent stromal cells, although these subconfluent stromal cells transfer growth factors to a chamber containing hematopoietic cells (Figs. 5A and 5B). The transfer of hematopoietic cells can be achieved by controlling the flow rate of the perfusion solution or other well-known methods. This allows you to get many different wells in the specified chamber, which are separated by the corresponding partitions, and after the seeding of one well, when the cells reach a confluent state, they will move to the next well, and then after sowing, they will similarly move to the next well. Another modification of the system is that after 8-12 weeks in the culture, hematopoietic cells act on new proliferating stromal cells. This is provided in one of several ways. According to the first method, the cell culture is treated for 3-5 minutes with EDTA (EDTA), which removes hematopoietic stem cells from stromal cells. Isolated cells are then transferred to a vessel with fresh medium, which may itself contain bone marrow stromal cells seeded 3-7 days before. This process is repeated every 8-12 weeks. And finally, small organic molecules or proteins, especially hormones, for example, a growth factor derived from platelets (at a concentration of 100-500 ng / ml), interleukin-1-alpha, tumor necrosis factor-alpha, or basic fibroblast growth factor, or others molecules mitogenic with respect to fibroblasts can be added to these cultures after 3-7 days. Such an effect on stromal mitogen-stimulating factors enhances the continuous proliferation of bone marrow stromal cells and their continuous synthesis of hematopoietic growth factors. Thus, it is possible to provide a continuous stage of subconfluent development of stromal cells.

 Continuous perfusion can also be used to separate healthy cells from cancer cells within a population of bone marrow cells. In this method, a radial flow cell is first used to determine the specific adhesion ability of healthy cells relative to cancer cells, and then a rectangular flow cell is used at predetermined flow rates that can provide shear stress sufficient to separate the cancer cells.

 The inventive method and apparatus also provide an opportunity for recycling of stem cells lost in the perfusion stream. The CD34 surface membrane marker provides separation of immature hematopoietic cells from mature cells. Thus, in the capture and recycling of cells that are related to CD34 +, stem cell loss can be avoided.

 Various methods can be used to capture and return the immature fraction of cells to the reactor. So, for example, you can enter the label of antibodies specific for CD34 in these cells, and then use antibodies against antibodies to collect CD34 + cells and their recycling in the reactor. Alternatively, you can select cells with a negative reaction, as a result of which you can ensure the removal of mature cells using antibodies against various markers associated with mature cells, such as antibodies to glycophorin, CD33, MO1, OKT3, CKT4, OKT8, OKT11, OKT16, OKM1, OKM5, Leu 7, Leu 9, Leu M1, Leu M3 and the like. There are many different antibodies for markers specific for mature cells of various hematopoietic differentiations, lymphoid, myeloid and erythroid, and these antibodies can be used to remove mature cells from effluent flows from the reactor, followed by collection of the remaining cells and their return to the reactor.

 Separation using antibody markers can be achieved in various ways, using standard methods individually or in combination, for example, panning, sorting cells with fluorescence amplification, binding of antibodies to various carriers, for example, the surface of polystyrene microspheres, metal microspheres and magnetic carriers, etc. Antibodies bind to surfaces that allow separation of adhesive from non-adhesive cells, or they label directly or indirectly into antibodies, resulting in a choice between labeled and unlabeled cells.

 Following the proposed methods, it is possible to significantly increase the life time of in vitro hematopoietic cells, usually hematopoiesis of human cells is ex vivo for about six months in culture, with the growth of granulocytes lasting for four months, and red blood cells for at least three months. In addition, hematopoietic progenitor cells are continuously formed throughout the growing period, leading to a 10-fold increase in the volume of net production of progenitor cells from the original volume.

 In addition to the above, adhering to the methods proposed in the present invention, it is possible to significantly increase the intensity of stem cell division, which makes possible the effective introduction of retrovirus-transfected genetic material. Genes introduced by the corresponding retroviral vector during the first two weeks from the time of infection can be expressed up to 10-30% in all progenitor cells and progenitor cells produced during further cultivation for four months in culture. The proposed methods, thus, ensure the successful delivery of the gene product into the highly proliferative human hematopoietic stem cell.

 In FIG. 1 is a schematic view of a perfusion chamber. The reactor 10 with a plate 12 located on top of the reactor and a plate 14 located at the base of the reactor are connected with bolts 16 to fix their position with wing nut 18. Three bolts are used to prevent skewing. The chamber 20 has three compartments, the middle compartment 22 contains a support matrix for stromal cells from stromal cells and bone marrow cells. The central part of the compartment 22 is separated from the upper section 24 and the lower section 23 by membranes or screens 28 and 30, respectively. For convenience, you can use a polysulfonate membrane or a stainless steel sieve, while the size of the sieve openings is sufficient so that cells can enter the central part of the chamber. An interphase separation partition can be installed in the chamber using an inner cylinder 27, which is divided into sections to provide mechanical support for the separation membrane. The upper section 24 and the lower section 26 may have different profiles and contain in cross section a pipeline or membrane, where the replacement of the nutrient medium and gases. Gases pass through a hydrophobic, for example, silicone tube, the size of which (and, consequently, the gas-liquid contact area) can be varied to provide the necessary number of incoming flows to meet the needs of the cell population whose metabolism occurs in the central part. The nutrient medium can be pumped, or it can flow by gravity from above and below the chamber through the opening 24, or enter through a pipe 34.

 If necessary, the upper and lower sections can be eliminated by replacing them with an external oxygenator. In this case, the separation membrane is fixed using a glass cylinder 36, which is installed in the cylindrical grooves of the plates 12 and 14, and the outer surface of the cylindrical groove is identical to its inner, which ensures excellent distribution of the incoming medium flow throughout the membrane. This geometry allows mixing of the flow from the end point of the inlets to stabilize the radial pressure. Such an arrangement is suitable for chambers that have relatively few cells; as a result, the oxidation process does not become restrictive.

 In FIG. 2 shows a schematic view of a circuit that connects a perfusion chamber to a reservoir with a nutrient medium, an oxygenator, a measuring (sensor) cell, and openings for supplying a sample and injecting a medium.

 An external source of fresh medium 50 is pumped by pump 52 into the medium tank through line 56, and the spent medium is removed through line 58 from tank 54 by means of pump 52 into the waste product tank for subsequent processing. The second pump 52 pumps the medium from the reservoir 54 through the pipe 64 through the hollow fiber of the oxygenator 66. The medium is sent through the pipe 68 to the first chamber of the bioreactor 70. For convenience, means have been installed for pumping the medium component 82, which transports the medium through the pipe 68 to the first chamber of the bioreactor 70. As this component can be investigated materials, additional growth factors and the like. The nutrient medium from the bioreactor 70 is directed through the Central part of the chamber 72 into the second chamber 74 of the bioreactor. From here, the medium flows through a pipeline 76 to sensors operating in real time to detect changes in the composition of the medium.

 For example, it is desirable that the ratio of glutamine to glucose be in the range of 1: 5-8 depending on the cell lines used, for example, a ratio of 1: 8 for transfected cells of the 3T3 line is preferably. Moreover, the ammonium concentration is preferably less than 2.0 mM, and the lactate concentration is preferably less than about 40 mM. When controlling the flows from the bioreactor, it is possible to modify the medium introduced into the bioreactor, change the partial pressure of oxygen and change the oxygen supply rate, add various components, or decrease or increase the speed.

 From the sensor elements, the medium is directed through a pipeline 80 by means of a pump 62 to a reservoir 54.

 Using the flow pattern described above, the medium in the side tank is slowly exchanged. Such an organization allows autonomous control of the frequency of medium replacement (external pump) and the flow rate through the oxygenator and perfusion chamber. The former is used for long-term regulation of variables in the composition of the medium and perfusion conditions, while the latter can be used to regulate the pressure of dissolved oxygen and the flow patterns in the chamber. The use of a small biocompatible mesh membrane allows the piston to move into the chamber, and this, in turn, allows precise control of the supply of growth factors and other special compounds that are desirable to add to hematopoietic and stromal cells in strictly defined quantities.

 After autoclaving the chamber of the feed circuit elements, said bioreactor is collected under sterile conditions. The medium can circulate along the side circuit and chamber for several days, while simultaneously controlling the degree of contamination. If sterile assembly is provided, then the central section of the chamber is inoculated either when the extracellular matrix is used purely or the pre-inoculated extracellular matrix substrate that contains stromal cells is used. After that, stromal cells either (1) are kept in the chamber for several days, while simultaneously regulating their metabolic parameters and / or growth factor sensitivity, and if the results are satisfactory, bone marrow is inoculated, or (2) bone marrow is inoculated immediately. In any case, the suspended cells are kept in the lower part of the central compartment of the chamber. Then the cells are superimposed on an additional extracellular matrix and the specified layer of cells is immobilized on the dividing septum. At this time, the chamber may tip over and the cell layer will move to the top of the indicated central section. With this configuration, maturing cells settle into the lower part of the central chamber, as they have lost their adhesive ability with respect to stromal cells. This feature is important to prevent damage to the stromal layer and / or less mature hematopoietic cells when exposed to mature cells. This feature also provides continuous, easier separation of mature cells.

 Stromal cells are mainly fibroblasts modified with one or more genes. The same or different cells can be transfected with genes depending on the particular choice of host cells, and the same or different cells can be used for many different genes.

 A wide variety of healthy cells or resistant cell lines can be used. However, not all cell strains have been found to be usable since the transformation of some cell lines can lead to excessive cell growth. It is desirable that the cells used are not neoplastic cells, and rather, immobilization on a substrate is necessary. For mammalian cells, it is not necessary that they are human or primed. Many non-transformed cells can be used in adhesive cell layers, including healthy adhesive cells of the human spleen and intact human thymus epithelial cells.

 Methods of transforming mammalian cells are well known, and there is a lot of literature on this subject, although only a few sources are provided in this description. These constructs can be used in a naturally occurring regulatory region of transcription initiation containing a promoter and an appropriate enhancer, or another region of transcription initiation, which may be inducible or unregulated, can be included.

There are many transcription initiation sites that have found application, and these include chromosomal promoters, for example, the promoters of mouse or human metallothionein-I and II, the actin promoter, etc. or viral promoters, for example, as an SU 40 early gene promoter, an SMU promoter, adenovirus promoters, promoters associated with LTRS retroviruses, etc. These promoters are readily available; they can be easily inserted into appropriate vectors that contain
polinkers for insertion of the transcription initiation site, as well as the gene under study. In other examples, expression vectors are used that contain a polylinker between the transcription initiation region and the transcription termination region, which enables the transmission of various signals related to translation messenger processing, i.e., a capping and polyadenylation site signal. When constructing a polygenic expression cluster containing regulatory regions and a structural gene, one or more restriction enzymes, adapters, polylinkers, in vitro mutagenesis, primer repair, resection, etc. can be used.

 An expression cassette is typically part of a vector containing a marker and one or more replication systems. The marker provides detection and / or selection of cells into which a polygenic expression cluster and marker are introduced. Various markers can be used, especially markers that provide resistance to toxin, especially an antibiotic. It is preferable to use the resistance of gentamicin, which provides resistance to the G418 mammalian host cell. As a replication system, one can use prokaryotic replication systems that allow for cloning at different stages, collecting individual components of a (polygenic expressing) cluster.

 When introducing a polygenic expression cluster into a host cell, any of well-known methods can be used, including calcium-linked DNA transformation, transfection, infection, electroporation, ballistic particles, and the like. Immediately after the modification of the host cells, they can be amplified in an appropriate nutrient medium containing a selective agent to select those cells that contain a marker. Surviving cells can then be amplified and used later.

 As host cells, the African green monkey CV-1 cell line, NIH-3T3 mouse cells, healthy human bone marrow fibroblasts, human spleen fibroblasts, healthy mouse bone marrow fibroblasts, and healthy spleen fibroblasts can be used. It should be emphasized that in some cases, depending on the selected vector and cell line, the cells go into a neoplastic state. Of great importance is the fact that the obtained transformed cells have the ability to adhesion, through which they provide immobilization on a substrate, for example, as a protein sponge, protein-coated membrane and the like.

 Immediately after constructing the vector for expression of the desired growth factor, it can be used to transform cells using any well-known method. The obtained transformed cells can be used to inoculate the substrates, which were mentioned earlier. These substrates can be introduced into the reactor or they can be present in the reactor during inoculation.

 The reaction mass can then be inoculated appropriately with hematopoietic cells. Hematopoietic cells can contain almost pure stem cells, a mixture of hematopoietic cells that practically does not contain mature hematopoietic cells of one or more differentiations, or a mixture that includes any of (or almost all) differentiations of the hematopoietic system at different stages of their maturation.

 Cells are cultivated by the almost continuous passage of perfusion solution through the reactor and the regulation of the levels of nutrients and growth factors included in the medium. For the most part, primary growth factors are maintained by stromal cells, as a result of which normal levels of growth factors are usually restored. Since conditioned supernatants have been found to be effective in the cultivation of hematopoietic growth factors, it is possible to provide a ratio of stromal to hematopoietic cells that maintains the growth factor at the proper concentration level in said reactor.

 Transfected stroma allows for the introduction of genes into human stem cells. In mice, retrovirus-mediated gene transfer to stem cells can be performed by pretreatment of 5-FV mice followed by growth of bone marrow maturing cells in WEHI conditioned medium containing IL-3 and GM-CSF (Lemischka) Cell (1986), 45: 917) . A synthesized stroma grown with a packaging defective cell line secreting the retroviral vector under study can be used to ensure efficient gene introduction into human stem cells. For example, human T cells can be made resistant to HIV infection by infecting stem cells with a retroviral vector containing the antisense HIV sequence under the control of the CDC2 regulatory sequence (Greaves, Cell (1989), 56: 979-986) tissue-specific gene expression in T cells. Such an effect should be exerted by the factor providing the cell line with retroviral packaging: this factor is absent in hematopoietic target cells. Once this virus is transferred to hematopoietic target cells, they immediately lose their ability to replicate.

 In FIG. 3A and B, a radial flow chamber 100 is shown having an inlet 102 and an outlet 104, and a chamber 106, where arrows 108 show the flow direction. Hematopoietic cells 110 are seeded in the stromal layer 112 in the chamber and cultured. The flow rate will be determined by cells that are capable of adhesion, and non-adhesive cells 114 exit through the outlet 104.

 In FIG. 4A and 4B, the culture chamber 120 is provided with an inlet 122 and an outlet 124. In FIG. 4B, inlet 122 comprises a valve system 128 that provides separate chambers 126 containing cells 110 and stroma 112 for cultivation and separation.

 In fig. 5A and 5B show culture chambers in which the septa 134, 136, 138 are removed during the cultivation process according to the following scheme: barriers 134 are removed at about 8-10 weeks, barrier 135 is removed at about 18-20 weeks, and the barrier 138 are removed at approximately 28-32th week.

 For a clearer understanding of the present invention, which is broadly described above, specific examples are presented to illustrate but not limit the invention in question, unless otherwise specified.

Illustrative Cell Separation and Staining Procedures
Ficoll Bone Marrow Cell Separation
1. A bone marrow sample is diluted in a ratio of 1: 4 in I-MDM medium maintained at room temperature (Dulbecco's medium, modified by the method of Claims, GIBCO, cat. N 430-2200).

2. Gently, a 5 ml layer of the diluted sample is transferred to 15 ml Ficoll-Paque at room temperature (specific gravity 1.077 g / cm ³ , Pharmacia; Cat. N 17-0840-02) in a 50 ml centrifuge tube.

3. Centrifuge at 700 · g (1800 rpm on a Beckmah) for 30 minutes at room temperature (20 ^° C.).

4. After centrifugation, most of the upper layer is removed (removing approximately 5 ml on top of the interphase), the interphase layer (bone marrow cells) is collected and washed three times in ice-cooled I-MDM medium as follows:
first rinse: 1400 rpm, 15 min, 4 ^o C;
second wash: 1200 rpm, 10 min, 4 ^o C;
third rinse: 1200 rpm, 10 min, 4 ^o C.

 5. After the third wash, the cells are suspended either in medium or in a balanced saline solution ... (depending on their further use), and the cells are counted after 1: 10 dilution of the cells in acetic acid (10 μl of cell suspension = 90 μl 2 % acetic acid in PBS, which allows only leukocytes to be counted, since red blood cells are lysed in acetic acid).

 6. Then the cells are suspended to the desired final concentration in the appropriate medium (for various applications).

Fluorescent staining of MY-10 positive bone marrow cells
Reagents
Standard buffer:
powder of dry Bacto-buffer DIFCO (Baxter, cat. N 2314-15GB) - 200 g,
10% NaN ₃ (sodium azide) - 20 g,
thermally inactivated amniotic calf serum (56 ^o C, 30 min) - 200 ml.

The volume was adjusted to 20 l in dd - H ₂ O, pH 7.15-7.25, stored at 4 ^o C. Suitable for use for 1 month
2% Paraformaldehyde solution:
paraformaldehyde - 10 g,
dd - H ₂ O - 500 ml,
10 n. NaOH (under a fume hood) - 8-20 drops,
powder dry Bacto-buffer DIFCO - 5 g.

Pour dd - H ₂ O into a 500 ml flask and stir in a hot cup heated to 60 ^° C in a fume hood.

 Add 10 g of paraformaldehyde.

 NaOH was added dropwise until the solution became clear.

 Add 5 g of DIFCO.

 The solution was cooled, and the pH was adjusted to 7.35-7.45 with 2N. HCl.

1. After counting the cells, they are washed once in standard buffer (100 rpm, 5 min, 4 ^° C).

2. Then the cells are suspended in standard buffer at a concentration of 2 · 10 ⁵ cells / ml.

 3. Two 50 μl aliquots of the cells are placed in 215 ml centrifuge tubes.

 4. 50 μl of a 1: 5-dilution of anti-HPCA-1 (antigen against human progenitor cells, Becton Dickinson, cat. N 7660, diluted 1: 5 in standard buffer) is added to one tube. 50 μl of a 1: 5-dilution of MIg (mouse IgG1 control, Becton Dickinson, cat. N 9040, diluted 1: 5 in standard buffer) was added to another tube.

 5. Then both tubes are incubated on ice for half an hour.

6. After incubation, the cells are washed twice in 5 ml standard buffer (1000 rpm, 5 min, 4 ^o C).

 7. After the second wash, cell debris is resuspended in 50 μl of a 1: 40-dilution of GAM-FITC (affinity isolated goat F (ab '), 2 anti-mouse IgG and IgM, adsorbed human Ig, fluorescein conjugated, TAGO, cat. N 4353, diluted 1:40 in standard buffer).

 8. Cells are incubated for 1/2 and 1 hour in the dark on ice.

9. After incubation, the cells are washed twice in 5 ml of standard buffer (100 rpm, 5 min, 4 ^° C), and each debris is resuspended in 100 μl of standard buffer + 100 μl of a 2% paraformaldehyde solution.

 10. Then the cells are analyzed for fluorescence using a flow cytometer. The percentage of positive fluorescence is% fluorescence in the anti-HPCA-1 sample minus% fluorescence in the MIg sample.

Bone marrow fluorescence staining for the selection of mature progenitor cells
Goals
The goal of this staining is to enrich the hematopoietic stem cells (the most primitive stem cells) by removing mature cell populations using a flow cytometer or magnetic microspheres. Almost always, some cells should be left as complete bone marrow cells (after separation by Ficoll) for staining for MY-10-positive cells in order to compare the effectiveness of sorting and determine the degree of enrichment.

1. Cells are divided into Ficoll-Paque, as described above. (0.5 × 10 ⁶ cells are isolated and divided into two parts for staining with anti-HPCA-1 / GAM-FITG and MIg / CAM-FITC).

2. After the third wash, the cell debris is suspended in a cocktail containing a monoclonal antibody (see the description of the manufacture of this cocktail) using 1 ml of a cocktail for 10 ⁷ cells, and the cells are incubated on ice for 1 hour.

3. Then the cells are washed three times in excess with ice-cold I-MDM medium (1000 rpm, 5 min, 4 ^o C).

4. After the third wash, the cells are suspended in a 1: 40 dilution of GAM-FITC (diluted in I-MDM, non-standard buffer) at a flow rate of 50 μl per 0.25 · 10 ⁶ cells and incubated on ice in the dark for 1/2 h .

 5. After incubation, the cells are washed three times in ice-cold I-MDM medium, and after the last wash, the cells are suspended in 2-4 ml of ice-cold I-MDM and kept on ice until sorting.

 6. Then the cells are sorted on a fluorescence flow cytometer to exclude the upper 85% fluorescence histogram. For better enrichment, sorting can be repeated twice.

 7. After sorting, cells are counted, washed, and an aliquot is stained for MY-10 positive cells (as described above) in order to determine the degree of enrichment compared with stained aliquots of complete bone marrow cells.

Selection for immature cells using magnetic antibodies
1. After performing stages 1-3, mature cells are sorted out in the procedure for fluorescent staining of bone marrow cells.

 Attention! Sodium azide is not part of any of the buffers.

2. The corresponding amount of magnetic goat anti-mouse Ig (Biomag: Collaborative Research; Cat. N 74340-50, 1 mg / ml, 5 · 10 ⁸ particles / ml) is washed three times in ice-cooled I-MDM medium at 1500 r / min, 5 min, 4 ^o C (for washing sodium azide, which was used as a preservative).

3. Resuspend the cell debris obtained after the third wash in the “Stage 1” in Biomag at a level of 50 Biomag / cell particles. (for example, 5 · 10 ⁷ particles are used for 1 · 10 ⁶ cells, and therefore 0.1 ml of Biomag).

 4. Cells are pelleted in a T-25 or T-75 tissue culture flask (depending on the number of cells) and incubated for half an hour on ice, shaking occasionally.

5. After incubation, the flask is placed on a flat magnet (supplied by Biomag) and fixed using rubber or adhesive tape, and incubated for 10-15 minutes at 4 ^o C.

 6. Place the magnet and flask upright and collect the supernatant.

 7. Steps 4-6 are repeated two more times.

 8. Cells were counted, washed once in ice-cold I-MDM, an aliquot was isolated for staining with MY-10 positive cells, and resuspended in appropriate media for future use.

I. Replacing the medium
Materials and methods
Cells: Human bone marrow cells were obtained from a heparinized aspiration biopsy taken from the iliac crest of volunteers who gave informed consent. Bone marrow was isolated by centrifugation in a density gradient Ficoll-Paque (Pharmacia, N 17-0840-02), and cells with low density (<1,077 g / cm ³ ) were collected and washed three times with Dulbecco's medium modified by the suit method (IMDM) . Between the second and third washes, cells were counted. Cells were then plated on 24-well tissue culture plates (Costar No. 3524) with double or triple duplication at 1, 2 and 5 · 10 ⁶ cells / ml at 322 μl per well.

Conditions for long-lived cultures: low-density cells were incubated in IMDM supplemented with 10% Laboratories Hyclone calf fetal serum, 10% horse serum (Hyclone Laboratories), 1% penicillin / streptomycin (Sigma, 10,000 units / ml penicillin G and 10 mg / ml streptomycin, cat. N P3539) and 10 ^-5 M hydrocortisone (17-hydroxycorticosterone, Sigma, cat. N H0888) in a humidified atmosphere containing 5% CO ₂ and 95% air. The cultures were treated with one of three media for exchange: 100% medium for daily replacement (7 / week), 50% medium for daily replacement (5.5 / week) or 50% medium for replacement twice per week (1 / week). Twice a week, while replacing the medium, 50% of non-adherent cells were removed from each culture well and counted using a hemocytometer.

 When the cells were removed for counting (2 / week), all media removed during the 3.5 / week and 1 / week cultures were stored for cell counting and fresh medium was fed into the wells. Of the 7 weekly cultures, half was retained for cell counting, and non-adherent cells were centrifuged and returned to the remaining half of the medium to be removed. Then, fresh medium was added to each well to replace the medium removed for cell counting. If cells were not removed for counting, then from each well from 7 / week. and 3.5 / wk cultures were removed 100% or 50% of the medium, respectively, the cells were centrifuged and returned to the original wells together with the added fresh medium.

 Methyl cellulose and morphological analyzes: once every two weeks, non-adherent cells removed for counting were plated in methyl cellulose in the presence of erythropoietin, GM-CSF and IL-3 and granulocyte macrophage colony forming macrophage units (CFV-GM) were counted. Aliquots of the removed cells were cytocentrifuged, Wright-Giemsa stained and differentiated cells counted.

 Statistical analyzes: the results of cell production over two weeks were expressed as mean ± cf. sq. osh from duplicate crops. The probability of significant differences between groups of cultures was determined by comparing the normalized values of the cumulative production of cells obtained from quickly replaced cultures (7 / week. And 3.5 / week.) In relation to the control cultures (1 / week.) Using the two-sided T-criterion . Statistical significance was considered at a 5% level.

results
Kinetics of the production of non-adhesive cells: the production of non-adhesive cells was evaluated as a function of the density of the cell inoculum (within 1-5 · 10 ⁶ cells / ml) and the metabolic rate. The rate of medium exchange varied from the exchange of one volume of medium per week (traditional culture of the Dexter type) to the exchange of the entire volume of medium 7 times a week. The number of cells collected twice a week was normalized by dividing by the number of cells inoculated into the culture.

 At each medium exchange rate, the curves of normalized values of the number of collected cells do not show a significant change depending on the inoculum density. Cell production for cultures supported by three modes of metabolism: 7 / week., 3.5 / week. and 1 / week., was the same after their normalization to the number of inoculated cells per culture. Comparison of the final cumulative production of cells at different inoculum densities did not show significant discrepancies for any exchange mode (p> 0.2 with a two-sided T-test for all pairs of samples).

 In contrast, the rate of medium exchange is very important for the speed and duration of cell production in these cultures. Cell production in cultures replaced at 1 / week. (control), 3.5 weeks. and 7 / week declined in the first few weeks. However, this difference in crop productivity became apparent after 3 weeks. After 3-10 weeks, cell production remained constant in 7 / wk cultures, constant at a lower level in 1 / wk cultures, but increased exponentially in 3.5 / wk cultures. After 10-12 weeks, cell production decreased in all cultures until its completion.

 The results obtained for 1 / week. -exchangeable cultures are equivalent to those that are usually observed in traditional Dexter-type cultures in a number of systems, whereas rapidly exchanged cultures are 3.5 and 7 / week. show increased cellular productivity compared to previously used methods of optimal cultivation. Cultures in which half of the medium was exchanged daily (3.5 / week) retained increased cell productivity for a longer time than the control (1 / week) or completely replaced daily culture (7 / week). After 3–9 weeks, the number of non-adherent cells harvested from 3.5 / week-exchanged cultures exponentially doubled every 2.1 weeks.

 Cell production at 3.5 / week. and 1 / week can be directly estimated by plotting the cell production curve at an exchange rate of 3.5 / week. as a percentage of crop production relative to crops with a 1 / week exchange regime. These comparisons showed that the duration of the initial phase of decreased cell production was the same for the two modes. However, after 3.5-18 weeks, the cell productivity of cultures with a mode of exchange of 3.5 / week. remains at a constantly higher level.

The proliferative potential of cultures can therefore be determined by their ability to produce cells after the initial phase of decline. Normalized number of cumulative cell production obtained after 3 weeks (Σ $\genfrac{}{}{0ex}{}{\text{n}}{\text{i}}$ = 7 'C _i / C ₀ ) does not depend on the density of the inoculum for medium exchange modes 7 / week. and 3.5 / week. The cell production data obtained for cultures with similar modes of medium exchange are quantitatively and statistically similar, and therefore they were averaged and combined (bottom panel) to obtain a larger statistical sample. The density-averaged cumulative cell production after 3.5–20 weeks was: 0.22 for 7 / wk, 0.40 for 3.5 / wk. and 0.15 for 1 / week.-crops.

 Thus, the increase in the rate of exchange of the medium from 1 / week. up to 7 / week contributed to an increase in cell production by about 60% compared with the productivity of traditional dexter-type cultures. As a result, the exchange rate of the medium is 3.5 weeks. yields an almost 3-fold increase in cell production compared to the productivity of cultures with a Dexter metabolic rate (1 / week). Statistical analysis of these data using the bilateral T-test showed a significant difference between cultures from 7 / week. and 1 / week -mode, as well as between cultures with 3.5 / week.- and 1 / week.-modes with a significance level of 5%. Thus, the medium exchange mode is 3.5 / week. contributes to increased cell production compared to the traditional 1 / week dexter mode.

The production of progenitor cells of granulocytes and macrophages
The analysis for the production of progenitor cells was carried out by reproducing experiments with this mode of perfusion of the medium and the given density of the inoculum (table 2). The perfusion rate of the medium had a noticeable effect on the number of granulocyte and macrophage precursor cells produced. The greatest duration of the production of progenitor cells was found in cultures with an exchange rate of 3.5 / week. These cultures produced precursors at a stable level after 4-18 weeks from the start of cultivation.

Optimum conditions for the production of precursor cells gave cultures with a medium exchange rate of 3.5 / week. and inoculation density of 5 · 10 ⁶ cells. / ml These cultures produced a significant number of progenitor cells up to the 20th week after the start of cultivation. Statistical analysis using the two-sided T-test showed that cultures with the indicated optimal metabolic rate (i.e. 3.5 / week) produce a significantly larger number of granulocyte and macrophage precursor cells after 8 weeks than 7 / week. - and 1 / week.-culture at all three inoculation densities at a significance level of 1%. The number of progenitor cells produced is an important factor as it is an indirect indicator of stem cell recovery. Precursor cells can be present in the culture only after a few weeks when differentiating from earlier cells, preferably stem cells that are still in culture. Thus, the obtained data suggest that a more physiological regime of rapid change of medium / serum and higher cell density may provide conditions under which a certain level of stem cell regeneration will be maintained for five months.

Morphology of non-adhesive cells: to determine whether there are qualitative differences between prolonged hematopoiesis, supported in 3.5 / wk cultures, from hematopoiesis in other cultures, non-adhesive cells were harvested at weeks 10-19, stained and typed morphologically. With the exchange modes 1 / week. and 7 / week the produced cells were mainly macrophages by the 15th week and later (table 3), which completely coincided with the results obtained in experiments conducted in other laboratories. In contrast, cultures with an exchange rate of 3.5 volumes of medium per week at a inoculum density of 5 · 10 ⁶ cells / ml produced granulocytes, as well as macrophages after 19 weeks. Thus, it is obvious that the indicated exchange mode and density of the inoculum are more effective for the restoration of granulopoiesis in vitro.

II. Replacement of the medium in combination with supplementation of the environment with hematopoietic growth factors
Materials and methods
Cells: Human bone marrow cells were obtained from heparinized aspiration biopsies taken from the bone marrow of the iliac crest of volunteers who gave informed consent in accordance with a procedure tested by the University of Michigan's Human Research Committee. Bone marrow was isolated by centrifugation in a density gradient Ficoll-Paque (Pharmacia), and cells with low density (<1,077 g / cm ³ ) were collected and washed 3 times with IMDM. Between the second and third washes, cells were counted. Cells were then seeded in 6-well tissue culture plates (Costar N 3406) or in 6-well plates coated with collagen (rat tail collagen type I, Biocoat. Collaborative Research Inc., Cat. N 40400) in duplicate 5 · 10 ⁶ cells / ml at 1.5 ml per well.

Culture medium: used IMDM medium (Gibo Laboratories, cat. N 430-2200) containing 10% fetal calf serum (Hyclone Laboratories), 10% horse serum (Hyclone Laboratories), 1% penicillin / streptomycin (Sigma, 10,000 units / ml penicillin G and 10 mg / ml streptomycin, cat. N P3539) and 10 ^-5 hydrocortisone (17-hydroxycorticosterone, Sigma, cat. N H0888).

 The result confirms the hypothesis that the conditions used for long-lived Dexter-type cultures are suboptimal, and that an in vitro culture that has a better approximation to hematopoiesis in vivo and is more effective for ex vivo bone marrow regeneration can be obtained.

 Physical characteristics: the exchange rate of the medium significantly affects the appearance of crops. At the 10th week in 7 / wk cultures there is a large number of lipocytes in the stroma, while 3.5 / wk cultures have very few fat cells, and 1 / wk cultures do not have fat cells at all. When the culture was depleted at the 26th week, the stroma of 7 / week-cultures consists of approximately 20-30% of fat cells, while 3.5 / week. cultures have only a few fat cells by this time. The distribution of adhesive colonies also varies with the perfusion rate of the medium. Adhesive colonies in 3.5 / wk cultures are more persistent than colonies in 7 / wk and 1 / wk cultures.

 Hematopoietic growth factors (HGH): it was found that due to the frequent replenishment of the culture by rapid change of medium, hematopoietic growth factors added to the medium at a concentration of approximately 1/20 contribute to the maximum formation of colonies in clonal assays 4. The following concentrations were used: 1 ng / ml 1-3, 1 ng / ml GM-CSF (Amgen Biological, Cat. N 13050), 0.1 u / ml Epo (Terry Fox Labs. Vancouver, Canada).

Analysis for the production of hematopoietic progenitor cells: non-adherent hematopoietic cells isolated from the culture were counted and plated in methylcellulose at a cell concentration of 1 · 10 ⁵ cells / ml or less. Then, GM-CSF and Epo were added to methyl cellulose at 20 ng / ml and 2 u / ml, respectively. These cells were plated in 24-well plates at 0.25 ml / well and incubated for 14 days at 37 ^° C. After this, colonies were counted using an inverted microscope, and colonies with more than 50 cells were counted as colony forming units of granulocytes and macrophages (CFU- GM), an erythrocyte burst-forming unit (BFU-E), or a colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM).

TBMC conditions: cultures were incubated at 37 ^° C. in a humidified atmosphere containing 5% CO ₂ and 95% air, and perfused with a daily change of 50% medium. During the first week of cultivation, 50% of all non-adherent cells were removed from the cultures twice a week during medium exchange, mononuclear cells were counted, and fresh medium was returned to the wells. On the remaining five days a week, when the cells were not counted, 50% of the medium was removed from each culture well and replaced with fresh medium, the removed medium was centrifuged, the medium was decanted from the cell pellet, and the cells were returned to their original wells.

 Statistical analyzes: the probability of a significant discrepancy between groups of cultures was determined by comparing the normalized values of the cumulative cell production from rapidly perfused cultures supplemented with hematopoietic growth factors with untreated control cultures using a two-sided T-test. Statistical significance was considered at 5%. At the same time, no statistical differences were observed between rapidly perfused LTBMCs cultured on plastic and on rat tail I collagen type I at a significance level of 5%. Therefore, the data for the plastic and collagen matrix were combined for presentation on graphs, and for statistical analyzes.

results
Kinetics of cell production in rapidly changing and supplemented with growth factor LTBMAc cultures
Since the first test was carried out to test the hypothesis that the duration and productivity of long-living bone marrow cultures (LTBMC) is limited by insufficient production of NSH, quickly replaced ex vivo bone marrow cultures were added to which IL-3 or Epo were added. In these cultures, 50% of the medium was removed daily and replaced with an equal volume of fresh medium supplemented with IL-3 or Epo. Then, the removed cells were centrifuged, the medium was decanted and discarded, the cells were resuspended and returned to the original cultures. IL-3 and Epo separately enhanced the cellular productivity of rapidly replaced LTBMCs. Cultures containing only one Epo initially had a high degree of cell production due to significant final erythroid differentiation. However, in the fourth week, erythropoiesis ceased, and the cell production rate decreased to the level of control cultures. IL-3 and Epo induced, on average, increased production of non-adherent cells compared to the control during 18 weeks of cultivation by 175 and 173%, respectively.

 The combination of growth factors has been shown to be more effective in increasing the rate of production of non-adherent cells. The highest degree of cell productivity was observed with the combination of IL-3 + GM-CSF + Epo. These cultures produced approximately 25% of the number of cells inoculated twice a week for the first six weeks, and had an average 4.8-fold increase in the production of non-adherent cells compared to the control within 2-8 weeks. The combination of IL-3 + GM-CSF yielded an average 3.5-fold increase in the production of non-adherent cells compared to a control for eight weeks. In separate experiments, the addition of either IL-6 or G-CSF to the combination of IL-3 + GM-CSF + Epo instead of the expected increase in the production of non-adhesive cells did not give a noticeable difference from cultures containing IL-3 + GM-CSF. In all cases, the stimulating effect on cell productivity induced by the addition of HGF was maximal for 0–8 weeks, although cell production was higher compared to the control during the whole cultivation time.

HGF combinations give high absolute values for non-adherent cells produced in fast-replacing LTBMC cultures. Culture productivity can be demonstrated by comparing the cumulative number of cells produced over a period of time (Σ $\genfrac{}{}{0ex}{}{\text{n}}{\text{i}}$ = 1, C _i ; C _i is the number of non-adherent cells collected by time i) relative to the number of inoculated cells (C ₀ ) by plotting the relationship (Σ $\genfrac{}{}{0ex}{}{\text{n}}{\text{i}}$ = 1, C _i , C ₀ ) versus time. If this ratio is greater than unity, then the culture produces more cells than was inoculated into it, and this culture leads to an expansion of the number of cells.

 The combination of IL-3 + GM-CSF + Epo promotes the induction of cumulative cell production, which is three times the number of inoculated cells. The degree of cell production was highest during the first six weeks of cultivation, during which time the culture produced approximately as many cells as were inoculated every two weeks. This maximum degree of cell production was 15% of the estimated in vivo degree of production of bone marrow cells, where 50% of myeloid cell mass was generated daily. The combination of IL-3 + GM-CSF gave more than 2-fold expansion of the number of cells, and at speeds comparable to those that gave the combination of IL-3 + GM-CSF + Epo for 3-7 weeks. Untreated, rapidly replaceable (daily 50% exchange medium) and slowly replaceable (50% medium exchange twice a week) control cultures that were not supplemented with HGF produced cells, the number of which was approximately 1- and 0.37-fold relative to the number of inoculated cells after 18 weeks, respectively. It is important to note that more than half of all cells removed from these incomplete cultures come from the first two samples, which indicates that many of these cells come from the original inoculum and the addition of cultures with HGF is necessary to induce a noticeable alternation of precursors and stem cells.

 Morphological analysis of non-adhesive cells: the addition of multiple amounts of HGF also leads to an increase in the diversity of myeloid cells produced in cultures. Control cultures produced non-adherent cells, which after three weeks were predominantly macrophages. The production of erythroid cells declined rapidly, and very few erythroid cells were detected after 5 weeks of cultivation. Cultures containing Epo (only Epo, IL-3 + Epo and IL-3 + GM-CSF + Epo) produced an unstable increase in the production of erythroid cells, with a high percentage (55-75%) of non-adherent cells accounted for erythroids in the third week of cultivation . If a combination of IL-3 + Epo + GM-CSF was present, then the cultures continued to produce erythroid cells for 16 weeks, with about 5-15% of non-adherent cells being typed as erythroid. Thus, the presence of IL-3 + Epo promoted active erythropoiesis.

 IL-3 ± Epo produced a population of non-adherent cells, which at the fifth week was predominantly composed of (60-70%) mature granulocytes (LG). The percentage of LG gradually decreased until at the 18th week it reached about 20%. Macrophage production increased accordingly. When GM-CSF was added to IL-3 ± Epo, a high percentage of LG remained for 18 weeks. Thus, the combination of IL-3 + GM-CSF promoted active granulopoiesis for 18 hours in culture, and the addition of Epo also supported erythropoiesis. Photomicrograms of the control and IL-3 + GM-CSF + Epo-supplemented cultures at week 5.5 showed a sharp increase in the density of the cultures and the diversity of the cells produced.

Kinetics of the production of non-adherent precursor cells
The production of progenitor cells increased with the addition of multiple amounts of HGF. The production of colony-stimulating units of CFU-GM macrophage granulocytes in untreated control cultures was long and constant over 18 weeks, consistent with earlier results obtained using rapidly perfused LTBMC without the addition of HGF. CFU-GMs produced in IL-3 + GM-CSF-and IL-3 + Epo ± GM-CSF-cultures are approximately 10 times higher than in control media for 3-5 weeks.

 It was previously reported that the production of erythroid burst-forming units in LTBMC rapidly decreases and ceases (Coutinho et al., Blood (1990) 75 11: 2118-2129). Quick exchange untreated control cultures showed a rapid decrease in BFU-E production, although low levels of BFU-E were produced for 17 weeks in the culture. The addition of one Epo did not significantly affect the number of BFU-E produced. IL-3 alone stimulates an easily transient increase in BFU-E production in 3-5 weeks. On the other hand, IL-3 + either Epo or GM-CSF induces a 10-20-fold increase in the levels of non-adhesive BFU-E compared to control levels during 3-5 weeks of cultivation.

III. Human Stem Cell Transformation
Materials and methods
Cells: Human bone marrow cells were obtained from heparinized aspiration biopsies taken from the bone marrow of the iliac crest of volunteers, who gave informed consent in accordance with a procedure tested by the University of Michigan's Human Research Committee. Bone marrow was isolated by centrifugation in a density gradient Ficolla-Paque (Pharmacia), and low density cells were harvested (<1.077 g / cm ³ ) and washed three times with IMDM. Between the second and third washes, cells were counted. For gene transfer experiments, bone marrow was obtained from patients with CD18 deficiency and their informed consent.

Selection of bone marrow cells with a negative reaction to differentiation (Lin ^- )
Mature mononuclear cells were isolated from the above cell preparations by incubating these cells with a mixture of monoclonal antibodies (MAb) after a third wash with IMDM. 10 ⁷ cells were incubated in 1 ml MAb cocktail on ice for 1 h, stirring lightly every 10-15 minutes. The MAb cocktail used is shown in Table 4.

Cells were washed 3 times with excess ice-cold IMDM and centrifuged at 4 ^° C. Corresponding amount of magnetic goat anti-mouse Ig (Biomag; Collaborative Research Corp., Cat. N 74340-50, 1 mg / ml, 5 · 10 ⁸ particles / ml ) washed 3 times in ice-cold IMDM and centrifuged. Cells were resuspended in Biomag at 50 ppm. and placed in a T-25 or T-75 tissue culture flask, then incubated on ice for half an hour, shaking occasionally. After incubation, the flask was placed on a flat magnet, attached to a magnet and incubated for 10-15 minutes at 4 ^o C. The magnet and flask were placed directly upright and the supernatant was collected. The described incubation with shaking, placing on a magnet, placing vertically and collecting the supernatant was repeated 2 more times. After this, the cells were counted and plated on 6-well tissue culture plates (Costar No. 3406).

The cultural environment. The experiments used IMDM medium (Gibco Laboratories, cat. N 430-2200) containing 10% fetal calf serum (Hyclone Laboratories), 10% horse serum (Hyclone Laboratories), 1% penicillin / streptomycin (Sigma, 10,000 units / ml penicillin G and 10 mg / ml streptomycin, cat. N P3539) and 10 ^-5 M hydrocortisone (17-hydroxycorticosterone, Sigma cat. N H0888).

 Hematopoietic growth factors. Used hematopoietic growth factors have been described above. The concentrations used were 1 ng / ml or 0.4 u / ml IL-3 (obtained from the Institute of Genetics, Cambridge, MA), 1 ng / ml GM-CSF (obtained from the Institute of Genetics, Cambridge, MA), 50 units / ml IL-1a (Genzyme Corp.), 0.1 U / ml Epo (Terry Fox Labs, Vancouver, Canada), 10 ng / ml MGF (mast cell growth factor, c-Kit ligand, Immunex Corp., Seattle, WA ), and 2.0 ng / ml Gibricin ([P1XY321] Immunex Corp., Seattle, WA).

Hematopoietic progenitor cell assay
Non-adhesive hematopoietic cells isolated from cultures during weekly sampling were counted and plated in methylcellulose at a concentration of 1-10 ⁵ cells / ml or less. Then, MGF, GM-CSF and Epo were added to methyl cellulose at 50 ng / ml, 20 ng / ml and 2 u / ml, respectively. After this, the colonies were counted using an inverted microscope, and colonies of more than 50 cells were qualified as GM colony forming units (CFU-GM), erythroid burst forming unit (BFU-E) or colony forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM) .

 Cell lines of retroviral producers.

 Retrovirus producer cell lines were obtained from Dr. Eli Gilboa at the Memorial Cloan Kettering Cancer Center, New York, NY. This cell line produces amphotropic viral particles that contain the NEO gene producing neomycin phosphotransferase that provides resistance to the mammalian neomycin G418 analogue. Both cell lines also produce retroviral particles that do not contain the genes necessary for the retrovirus, so cells infected with this retrovirus cannot produce the infectious virus themselves.

 The packaging-defective SAX-containing cell line is a 3T3-based cell line that contains a modified Moloney mouse leukemia virus (MoMuLV). SAX proviruses contain the NEO gene, the SV40-activated adenosine deaminase gene, at the XhoI restriction site. SAX proviruses also contain an Ψ region that does not contain the gag (capsid protein), pol (reverse transcriptase), and env (envelope protein) genes. This second retroviral particle contains a duplicate of foreign DNA, and these retroviral particles were designated DC-29 (duplicate 29th clone). The DC-29 proviruses contain 2 copies of the NEO gene and another retroviral and foreign DNA in the 3T3 cell line.

 For CD18 experiments, Psi-Crip amphotropic packaging-defective cells infected with a retroviral vector containing human whole were used (CD19-cDNA, Wilson et al., Science (1990) 248 1413-1416). In this retrovirus, complete human CD18 cDNA was cloned into a BamHI site of a vector that expresses a recombinant gene from heterologous sequences located in the 5 ′ region of the chicken β-actin gene designated BA-CD18. Sequences from 5 ′ to the early cytomegalovirus gene (IE) of the human cytomegalovirus were subcloned with PUC19, and the region containing the IE enhancer sequences was deleted starting from XhoI (from polylinker and NcoI (-220 gene IH fragment). Synthetic linkers were used to convert NcoI- site to the XhoI site, and the modified fragment was cloned into the unique XhoI site of BA-CD18, located in the region from 5 'to the β-actin promoter. This new vector was designated CMV-BA-CD18.

Retroviral Particle Production
SAX retroviral particles were obtained from the laboratory of Dr. Clay Smith Eli Gilboa Laboratory in the form of frozen and stored at -80 ^o C solutions of viral supernatant. Retroviral particles DC-29 and CD18 were produced by growing lines with defective DC-29 and CD18 virus packaging to a state close to continuity in a T-75 flask, replacing the whole medium, incubating cells for 12-15 hours, and then collecting medium containing viral particles. The virus-containing supernatant was then centrifuged to remove cells with viable packaging, after which the medium was removed and frozen in aliquots at -80 ^o C.

LTBMC with a supernatant containing SAX retroviruses, DC-29 retroviruses or CD18 retroviruses
The cultures were incubated at 37 ^° C. in a humidified atmosphere containing 5% CO ₂ /95%. During the first two weeks of culture, two-thirds of the medium (1 ml) was removed daily from each culture well, and the medium was replaced with an equivalent volume of medium containing HGF (0.85 ml) and a supernatant containing the virus producing cell (0.15 ml) . The medium containing the retroviral supernatant was thawed immediately prior to use, and if the medium must be used urgently, it was placed on ice in a refrigerator. The medium removed from the cultures was centrifuged, the medium was decanted, and the cells returned to the original wells.

LTBMC co-cultured with a SAX retrovirus packed cell line
The cell line with SAX-retroviral packaging was cultured to approximately 10% continuity in T-25 flasks (Costar, No. 3056), and then irradiated at 2000 rad. Hematopoietic cells obtained as described above were added to the irradiated virus producing cells and cultured with a daily change of 50% of the medium for 2.5 weeks, all cells being returned to the wells after changing the medium. After 2.5 weeks of cultivation, a 0.5 mM EDTA solution was added to the flasks in order to remove the hematopoietic cells that left the stroma. These removed hematopoietic cells were added to 3 wells of a 6-well plate with freshly trypsinized cells (1000 per well) of bone marrow fibroblasts.

Sampling LTBMC-infected:
Starting from the second week of cultivation, after the addition of retroviruses and the termination of co-cultivation, 50% of the medium was replaced daily in the cultures, and non-adhesive cells in the replaced medium were removed once for analysis. Non-adherent cells were removed from the cultures during daily medium exchange, mononuclear cells were counted, and fresh medium was added to the wells. On the remaining six days of the week when the cells were not counted, 50% of the medium from each culture well was replaced with fresh medium, the removed medium was centrifuged, after which the medium was decanted from the cell pellet, and the cells were returned to their original wells.

Retroviral infection test
The original bone marrow inoculum was plated at 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mg / ml G418 to construct an inhibition curve in order to determine the concentration of G418 at which the post-infected cells were cultured. Cells isolated from cultures were seeded in methylcellulose with G418 at concentrations of 0.0, 0.8 and 1.6 mg / ml. After two weeks, the number of colonies of the precursor cells in methylcellulose was counted. Separate colonies were then harvested from methyl cellulose and analyzed for the presence of retroviral DNA using polymerase chain reaction (PCR).

 Statistical analyzes: the probability of a significant discrepancy between groups of cultures was determined by comparing the cumulative amounts of produced cells from the experimental samples with the corresponding control cultures using a two-sided T-test. Statistical significance was considered at 5%.

results
Retroviral infection using SAX retroviruses
Kinetics of cell production in LTBMC infected with SAX retrovirus.

 The production of cells in cultures infected with a retrovirus is an indicator of the probability of infection with a retroviral infection, and therefore can be used as a parameter to measure the level of infection. The integration of the retrovirus into the genome of the target cell is likely to occur during cell division. For this reason, increased culture productivity increases the likelihood of stem cell mitosis, and therefore increases the likelihood of retroviral infection. The highest level of productivity occurs in cultures containing the virus supernatant and supplemented with IL-3 + GM-CSF and IL-3 + GM-CSF + IL-1α, which produce an increasing number of cells during 4 weeks of cultivation. LTBMCs cultured in conjunction with cells packed with SAX virus produced a greater number of cells than cultures supplemented with supernatant in 2 weeks, although cell production decreased after two weeks.

Retroviral infection assay in LTBMC supplemented with supernatant containing SAX virus
The percentage of surviving progenitor cells at high [G418] ranged from 2 to 50% in cultures supplemented with IL-31GM-CSF or IL-3 + GM-CSF + IL-1α during the first 4-6 weeks of cultivation. After 10 weeks of cultivation (8 weeks after the end of the virus addition), 43% of the number of progenitor cells that were capable of cloning into hematopoietic colonies survived when exposed to G418. This suggests that these progenitor cells had acquired resistance to G418 due to the content of the G418 resistance gene transferred by the retrovirus to stem cells present in the culture during the initial 14-day infection period. Quick-change cultures in which HGF was not added had an average of 12% surviving progenitor cells at high [G418] from 8 to 11 weeks of cultivation. After completion of cultivation, after 11 weeks, the stromal layer of cultures with added IL-3 + GM-CSF was trypsinized, and 17% of the progenitor cells were associated with the surviving stroma at high [G418]. This suggests that a significant percentage of adhesive progenitor cells was also infected with the SAX virus.

Analysis of retroviral infection in LTBMCs cultured in conjunction with irradiated cells with SAX virus packaging
The percentage of progenitor cells surviving in G418 when co-cultured with irradiated SAX cells varied between 0 and 36%. Cultures in which HGF was not added produced CFU-GM, which survived at high [G418] only for 4-7 weeks. After the 7th week, CFU-GMs that survived at high [G418] were not produced in these cultures, suggesting that the stem cells were very slightly infected or not at all infected. LTBMCs supplemented with IL-3 + GM-CSF and cultured together with irradiated SAX cells for 2.5 weeks produced a high percentage of CFU-GMs that survived at 0.8 mg / ml G418 on the 4th, 5th and 8th week. However, at week 10, CFU-GM production in these cultures plummeted due to the resistance of CFU-GM to G418. This suggests that in these cultures, stem cells are slightly infected or not infected at all, or that stem cells may differentiate or die.

Retroviral infection using retrovirus DC-29
Kinetics of cell production in LTBMC infected with DC-29 retrovirus
The number of cells produced in cultures infected with a supernatant containing DC-29 retrovirus is HGF-dependent. Cultures supplemented with IL-3 + GM-CSF + Epo produced 1.5-4 x 10 ⁶ cells, based on a weekly assessment over 10 weeks of cultivation. Cultures supplemented with IL-3 + GM-CSF + Epo + MGF were more productive, and cultures supplemented with Gibricin + Epo gave the highest level of cell production. It is interesting to note that control cultures supplemented with IL-3 + GM-CSF + Epo, but to which DC-29 retroviral supernatant was not added, were less productive than similar cultures supplemented with DC-29 retroviral supernatant. Cell production in IL-3 + GM-CSF + Epo-, IL-3 + GM-CSF + Epo + MGF-, and gibricin + Epo-cultures (with virus) was significantly higher than in the control culture (IL-3 + GM-CSF + Epo, without virus addition), with significance levels of 5, 1 and 1, respectively. Some of the increased cell production in cultures supplemented with a retroviral supernatant may be due to the presence of growth factors such as MGF (c-Kit ligand), which is known to be produced by a 3T3-based defective packaging cell line.

Retroviral infection assay in LTBMC supplemented with DC-29 retroviral supernatant
The efficacy of a retroviral infection was assessed using the survival rate of CFU-GM at 1.6 mg / ml G418, a concentration that killed all bone marrow cells before retrovirus infection. In all infected cultures (see table 5), the average percentage of survivors at week 8 (6 weeks after infection) of CFU-GM was high.

 As it turned out, the addition of MGF to the combination of IL-3 + GM-CSF + Epo contributed to an increase in infection efficiency by 8-10 weeks of cultivation, with one of the cultures containing 7.7% of G418-resistant colonies. Data obtained at week 8 suggest that cultures to which Gibrikin + Epo were added have a high infection rate, although data from week 10 indicate no infection. Regarding the data obtained for the 10th week, it should be noted that a decrease in the number of CFU-GM isolated from several cultures to a very small amount or to the complete absence of CFU-GM should be expected at an infection level of approximately 10%. In addition, a concentration of 1.5 mg / ml G418 represents an exceptionally high dose of the antibiotic, which is obviously quite sufficient to suppress the effectiveness of the transfected resistance gene for G418 at even very small suboptimal levels. Thus, the efficiency of the hematopoietic stem cell transfer gene in these cultures in some samples was at least 7.7%, and possibly 45% or higher.

Kinetics of the production of non-adherent precursor cells
Since the analysis of retroviral infection in these experiments depends on the evaluation of the progenitor cells in order to make a conclusion about the presence of stem cells, infection and cell changes, it is very important to evaluate the effect of HGF on cell production in culture. In addition, in this experiment with retrovirus, MGF and Gibricin were used in combination with other HGFs. Both MGF and Gibricin were not used in rapidly perfused HGF-supplemented LTBMCs, and therefore their effect on hematopoiesis was necessary.

 The production of progenitor cells was very dependent on the addition of HGF. The number of CFU-GM isolated from cultures is shown in table 6.

 Every second week, samples taken from the cultures were evaluated on a CFU-GM, and unvalued values were determined by linear interpolation between the two data points. Known and interpolated values were summarized to give an approximate value of the total number of CFU-GM isolated from the culture.

 The addition of a retroviral supernatant contributed to a 2.2-fold increase in progenitor cells compared to the cases when this addition was not done. The number of cultures isolated to which the retroviral supernatant and IL-3 + GM-CSF + Epo + MGF or Gibricin + Epo were added was 4.2 and 3.8 times higher than the number of CFU-GM from uninfected control cultures, respectively. The number of CFU-GM removed was statistically greater at a 1% significance level in all virus-supplemented cultures than the number of inoculated CFU-GM.

The production of progenitor cells in culture
The ratio of populations regarding the distribution of CFU-GM shows that the addition of MGF or Gibricin to cultures supplemented with IL-3 + GM-CSF + Epo gives a significant positive effect on the CFU-GM pool. The addition of MGF to the combination of IL-3 + GM-CSF + Epo contributes to a 1.9-fold increase in isolated CFU-GM, and the differentiation is 0.5-fold compared with similar cultures to which MGF was not added (see table 7).

 Every second week, samples taken from the cultures were evaluated on a CFU-GM, and unvalued values were determined by linear interpolation between the two data points. Known and interpolated values were summarized to give an approximate value of the total number of CFU-GM isolated from the culture.

 The combination of Hybridin + Epo had a very significant effect on the production and differentiation of CFU-GM. Gibricin + Epo yielded a 1.8-fold increase in the number of CFU-GM removed and a more than 3-fold increase in CFU-GM differentiation compared to previously optimal cultures supplemented with IL-3 + GM-CSF + Epo. In addition, Gibrikin + Epo facilitated the production of almost twice as many granulocytes and macrophages as was the case with the combination of IL-3 + GM-CSF + Epo + MGF. This suggests that Gibrikin is a strong inducer of the line of differentiation of granulocytes and macrophages.

 Analysis of neutrophils produced from stem cells infected with CD18 coding retrovirus.

 Bone marrow with CD18 deficiency was enriched with early hematopoietic cells, as described previously, and then cultured for 14 days with daily replacement of 50% medium with supernatant supplemented with 1.0 ng / ml / day GM-CSF and 1.0 ng / ml / day IL-3 and 40 u / ml / day IL-1α and line CD18-retroviral producer. Starting on day 15, cells were cultured under the same conditions, but without the addition of a retroviral supernatant. Non-adherent cells were removed from the cultures weekly and analyzed for the presence of CD18 cell surface by flow cytometry using biotinylated anti-CD18 monoclonal antibodies using standard methods (Updyke et al., Meth. Enzymol. (1986) 12: 717-725). CD18 deficient bone marrow cells were not able to express cell surface CD18 protein in this assay, while neutrophils and monocytes, which increased in cultures with retroviral infection, expressed cell surface CD18. In triple duplication cultures, expression of CD18 cell surface expression was 3.5% / 5% / 2% at week 6 and 11% / 28% / 3% at week 11. Since neutrophils and monocytes present in cultures after 11 weeks contribute to the formation of CFU-GM progenitor cells only 10-14 days earlier, this indicates that hematopoietic stem cells are sufficiently and stably transfected with recombinant retrovirus during the first two weeks cultivation.

 To interpret the results, it is important to understand that although some groups exhibit the transfer of the retrovirus gene into human hematopoietic progenitor cells, however, gene transfer to human hematopoietic stem cells has not been demonstrated. The rapid decline in cell production in traditional slowly perfused human LTBMCs does not allow the degree of infection to be determined due to the lack of progenitor cells necessary for analysis.

 Retroviral infection in the present studies was first assessed by culturing cells removed from LTBMC in methyl cellulose in the presence of the mammalian cell antibiotic G418. Precursor cells infected with SAX or DC-29 retrovirus and expressing the NEO product must be able to survive and colonize at high concentrations of G418, while uninfected cells must die at high concentrations of G418. In addition, for the production of progenitor cells that survive at high concentrations of G418 6 or more weeks after the end of the virus addition, it is necessary that these cells are just differentiated from more primitive cells (stem cells), and therefore it can be assumed that stem cells were infected. Similarly, for CD18 expression on the surface of neutrophils and monocytes produced during the 11th week of cultivation, the primitive hematopoietic stem cell must be infected during the first two weeks of cultivation, since all mature cells, precursors, and clonogenic precursors present during the period infection, died after 4-5 weeks of cultivation.

 The retroviral infection assay used in this study may underestimate the percentage of cells infected with a retrovirus due to insufficient expression of the NEO gene product. It was shown that insufficient expression of the product of the transferred gene creates certain inconveniences when using models of humans and primates. Therefore, in this study, the estimate of the percentage of progenitor cells infected as described above is probably underestimated.

 The percentage of progenitor cells that survived at high [G418] was approximately 40% at week 10 in cultures infected with SAX retroviral supernatant supplemented with IL-3 + GM-CSF ± IL-1α. These preliminary results showed that a high percentage of stem cells were infected in cultures supplemented with a retroviral supernatant during the first two weeks of cultivation.

 The percentage of progenitor cells infected with DC-29 retrovirus was high (0-21%) in IL-3 + GM-CSF + Epo + MGF and Gibricin + Epo cultures during the first four weeks after the end of virus addition. This high infection rate of the progenitor cells is likely due to direct infection of the precursor and primitive cells with retroviruses. The percentage of progenitor cells surviving in high concentrations of G418 decreased 4 weeks after completion of virus addition, but 2 weeks later recovered to 0-22% of surviving cells.

 An experiment using DC-29 in the production of G418 resistant progenitor cells likely provides an underestimated percentage of the progenitor cells infected with DC-29 retrovirus. The high concentration of G418 used to select infected colonies (1.6 mg / ml G418) is twice the concentration used in the experiment with SAX infection, and high levels of expression of the NRO gene product, neomycin phosphotransferase, are required for survival.

 It is interesting to note that in LTBMC, to which a supernatant was added containing SAX or DC-29 retrovirus, HGF, IL-3 + GM-CSF ± Epo, IL-1α, or MGF, or a combination of gibricin + Epo, the percentage of cells precursors surviving in the presence of G418 increased 6-8 weeks after completion of virus addition. Although the mechanism for increasing the percentage of progenitor cells surviving in the presence of G418 is not known in the later stages of cultivation, it can be assumed that stem cells that were infected during the first two weeks of cultivation become more active during cultivation. This fact contributes to an effective increase in the percentage of cells surviving in the presence of G418. As another explanation for this phenomenon, it can be assumed that the expression of the NEO gene product increases in progenitor cells that are late produced in culture due to their differentiation from stem cells that have a different and more expressed region of integration than precursor cells transfected directly during initial period of infection of the culture. Therefore, the high level of survival of progenitor cells in the late cultivation period, although there may be several reasons, however, it is most likely due to the fact that stem cells were infected in these LTBMCs.

 Summarizing the above, it can be argued that the data obtained indicate that under the cultivation conditions disclosed in this application, hematopoietic stem cells proliferate in these cultures, which allows the introduction of genetic material transferred through retrovirus into these cells. Precursor cells were constantly and actively produced from these stem cells, and many of these precursors contained and expressed transfected genes. The data obtained showed that genetically modified human hematopoietic stem cells were present and proliferated in these cultures.

IV. The experiments
I. Construction of transformants
Human growth factor GM-CSF (Wong. Science (1984) 228: 810-815) was administered a eukaryotic expression vector. hGM-CSF cDNA (EcoRI-AhaIII fragment, approximately 700 bp) was cloned into the EcoRI-PstI fragment of pSP65 (Melton, Nucl. Acids. Res. (1984) 2: 7035-7056). The resulting plasmid was designated P65GM-CSF. The mouse metallothionein promoter (Glanville, Nature (1981), 292: 267-269) was digested with EcoRI and BgIII, and a approximately 2 kb fragment containing this promoter was inserted into the EcoRI-BamHI fragment of pSP65 to obtain p65T. The pMT GM-CSF plasmid was then constructed by digesting pSP65GM-CSF using EcoRI, filling the sticky ends with a Maple DNA polymerase 1 fragment, and then digesting the resulting linearized DNA with HindIII to isolate a 700 bp fragment containing the coding GM-CSF area. This fragment was subcloned into the SalI - filled HindIII site p65MT. Then a 2.7 kb fragment containing the metallothionein promoter and GM-CSF coding region was isolated and introduced into pSV2neo (Southen and Berg. J. Mol. Appl. Genet (1982) 1: 327), from which the SV-40 promoter was removed . This led to the fact that the SV-40-poly A-signal was below (5 '--->3') from the GM-CSF coding sequence.

 The neomycin-resistant gene that reported antibiotic resistance to gentamicin (G418) was taken from pSV2neo by isolating the PvuII-EcoRI fragment at about 3 kb and EcoRI linkers were introduced into the PvuII site. The neo-resistance gene with EcoRI ends was subcloned into the EcoRI site of the GM-CSF-expressing plasmid to construct the MTGM-CSFneo plasmid.

 Plasmid MTGM-CSFneo alone and as a cotransfectant with a plasmid (Yang, Cell (1986) 47: 3-10) encoding the gibbon (monkey) gene IL-3 under the control of the SV-40 promoter and poly-A site was transfected by electroporation of linearized DNA into the African green monkey monkey CVI cell line and NIH 3T3 mouse cell line. Transformants were selected by selection in a medium containing 500 mg / ml G418, isolated and screened for the production of GM-CSF or IL-3 using bioassay of supernatants using AML-193 cells (Adams et al., Leukemia (1989) 3: 314) . Some positive lines were then used as a stroma for bone marrow cells in a Dexter culture.

 In addition, normal mouse bone marrow cells were transfected with the above plasmids using the Okayama calcium / phosphate method (Chen. Mol. Cell. Biol. (1987) 7: 2745-2752) and it was found to efficiently express the introduced genes.

The secretion of GM-CSF and IL-3 by transfected fibroblasts was investigated. The supernatants of a 72-hour serum-free culture were obtained from NIH-3T3 cells and analyzed for hGF secretion by ³ H absorption on target cells, inhibited by neutralizing rabbit anti-GM-CSF or anti-IL-3 antibodies. Proliferation induced by 20 mg / ml GM-CSF was set at 100 units. GM-CSF, and proliferation induced by 10 ng / ml IL-3 was set to 100 units. IL-3. These co-transfected cells produced about 35 units / ml GM-CSF and about 57 units / ml IL-3.

II. Perfusion chamber
The perfusion chamber was a glass cylinder with Derlin caps, which allow autoclaving without deformation and biocompatibility. These covers have cylindrical grooves into which a glass cylinder is inserted. A sealing ring is placed at the bottom of the groove to seal the lumen in the chamber. These covers have several openings into which Leuer fittings (Leuer Lok) are inserted, and medium and gas supply lines are inserted into them, as well as an extension pipe leading to the central section of the chamber for sampling adhesive and / or non-adhesive cells. These covers are attached with three long bolts, spaced 120 ^o apart and located outside the glass cylinder, with ring gaskets and nuts used for tight assembly.

 This camera is attached to the side tank. This loop contains a pump, a real-time sensor chamber, an oxygenator and, in addition to a side reservoir, sampling and injection holes. The medium in the side tank is then slowly exchanged using a separate pump. This configuration allows you to separately control the rate of exchange of the medium and the flow rate through the oxygenator and perfusion chamber. The oxygenator is used to control the long-term exchange of medium composition and perfusion, and the perfusion chamber can be used to control the pressure of dissolved oxygen and the flow structure in the chamber. The use of a polysulfonate membrane with small holes allows for a piston flow regime and precise control of the supply of growth factors and other specific compounds that need to be introduced into the bioreactor in very precise quantities.

 Transfected stromal cells are seeded either on top of the crushed collagen sponge layer, or they are seeded on one side of a 5 μm porous polycarbonate filter precoated with collagen, which allows stromal cells to bind to the filter for several hours. Cells are cultured in an appropriate nutrient medium until the cells reach a state of continuity on one side, and cytoplasmic ejections are carried out through the pores. Then, bone marrow cells were sown on the other side of the membrane, and stem cells bind to the implanted cytoplasmic ejections passing through the pores.

 After autoclaving the chamber and loop components, the reactor was mounted under sterile conditions. Then the medium was passed through the side loop and the chamber by circulating it for several days, and monitored for signs of contamination. The central part of the bioreactor was then inoculated with either one extracellular matrix or a pre-inoculated extracellular matrix substrate that contained stromal cells. Stromal cells can then be kept in the chamber for several days, and their metabolic productivity and / or growth factor reactivity is monitored, and if the results are satisfactory, the bone marrow is inoculated or the bone marrow is seeded immediately. In any case, the cell layer was kept at the bottom of the central section of the perfusion chamber.

 Cells were seeded on the extracellular matrix and the cell layer adhered to the substrate. When using the membrane, the chamber can be turned upside down and the cell layer will be located on the flow of the central section. With this configuration, maturing cells settle to the bottom of the central chamber, since they lose their adhesion to the stromal layer. Non-adherent cells are then harvested by means of a constant cell flow caused by the pressure of the perfusion medium and guided into the outlet pipe.

In a normal cycle, the chamber is inoculated with NIH-3T3 cells on the first day of the experiment, plating on a substrate from a crushed collagen sponge. During the first 40 days, perfusion rates and other parameters of the operation are adjusted. On the 40th day, stationary conditions are reached, which are maintained for about 20 days. On the 64th day, the chamber is seeded with 33 · 10 ⁶ bone marrow cells. During the first 10 days, the number of harvested cells decreases until it reaches a steady level of about 7-8 · 10 ⁵ cells every three days. Flow cytometric analysis showed that the constant fraction, about 20% of the collected cells was HLA-DP-positive. On day 90, the pump was turned off and the pH dropped below 6.9 overnight. When the perfusion rate was restored, the production of non-adhesive cells was restored and approached the previous stable level of production if bacterial contamination took place. At this stage, the study was completed.

 The results showed that the perfusion chamber allows ex vivo hematopoiesis, hematopoiesis can be restored ex vivo after a drop in pH, and glucose concentration data indicate that hematopoietic cells first develop aerobically on glucose because glucose concentration decreases after inoculation, and the concentration lactate does not increase, which indicates that oxygenation is limited. Obviously, glucose / lactate (anaerobic) metabolism is primarily due to the stromal layer IH-3T3. Similarly, the concentrations of glutamine and ammonia reach levels before inoculation after the number of hematopoietic cells decreases, which indicates that the uptake of glutamine by bone marrow cells is much less than the uptake of the stromal layer.

III. Metabolism Control
The rate of absorption and formation of glucose and lactate, as well as glutamine and ammonia was determined for transfected NIH-3T3 cells. (Used medium: IMDM + 20% fetal calf serum). An increase in glucose uptake was observed only for daily fed T-flasks, while fed cultures showed the same slow decrease in glucose uptake. Cultures in which the exchange rate from 50% daily was changed to 100% daily on the 18th day of cultivation showed a direct increase in glucose uptake with the same tendency observed for cultures with 100% daily exchange from the first day cultivation. The rate of lactate production was similar, since their ratio was mostly constant (lactate / glucose = 0.9, which indicates a predominantly anaerobic stromal metabolism).

The concentrations of glutamine and ammonia were the same in nature as in the case of glucose-lactate metabolism. Using values adjusted for the chemical decomposition of glutamine at 37 ^° C, it was shown that the ratio of glutamine to glucose absorption levels remains constant, about 1: 8 = glutamine: glucose. This predicted optimum ratio varies depending on the rate of oxygen uptake (this ratio decreases with an increase in its optimal absorption level).

 Similar conclusions were made regarding data for glucose / lactate metabolism obtained from normal stromal bone marrow fibroblasts. Under conditions of frequent environmental changes, cultures are first anaerobic with constant high and rapidly achieved levels of lactate. With a more frequent change of environment, cellular metabolism becomes faster, with an increase in glucose uptake and lactate production. After correction of data for spontaneous chemical decomposition, no significant glutamine uptake was observed. Studies with 3T3 cells and normal bone marrow cells showed that the cells continued to divide and grow rapidly at a serum / medium exchange rate that exceeded, obviously, the critical regime of medium change.

 To assess the relative importance of the speed of serum perfusion with respect to the speed of perfusion of the nutrient medium, the following experiments were carried out: 1) one series using T-flasks with a daily exchange of medium containing 20% serum, 2) two series with T-flasks, where in one series, 20% of serum was used, and the medium was changed every other day, and in another series, 10% of serum was used with daily medium exchange, 3) two series with T-flasks, where one contained 10% serum and the medium changed every other day, and in another contained 5% serum daily about exchange of medium; 4) two series with T-flasks, where one contained 5% serum with medium exchange every other day, and the other contained 2.5% serum with daily medium exchange. The serum metabolic rate in each group was the same, while the metabolic rate of the nutrient medium varied. The results of these series of experiments showed that the serum metabolic rate is a critical parameter. Although for experiment (1), glucose uptake increased, and on day 4 it leveled off to approximately 9.5 mM per day, however, in all other cases, glucose uptake was lower than the initial glucose uptake of group (1) and fell approximately linearly regardless whether a double amount of serum was used, and whether the medium was changed daily or every other day. The result confirms the conclusion that the perfusion rate of serum or one or more of its components is a critical parameter that affects the metabolic growth of stromal cells.

 From the above results, it is obvious that hematopoietic cells can be successfully cultured in a bioreactor. Stromal cells can be obtained from homologous or heterologous sources, and these stromal cells can be transfected with genes to provide important growth factors. Thus, to maintain cell growth, it is not necessary to add serum to the medium. Obtaining stromal cells that attach to the substrate so that the hematopoietic cells can be separated from the stromal cells allows you to constantly collect hematopoietic cells for use. By appropriate selection of combinations of factors, hematopoietic cells with a specific direction of differentiation can be cultivated. In addition, if necessary, stromal cells can replenish the stocks of transfecting viruses for introducing genes into hematopoietic cells.

 All publications and patent applications cited in this application are introduced into its description by reference, unless otherwise specified.

 Obviously, various changes and modifications may be made to the present invention, however, without departing from the spirit and scope of the invention as set forth in the claims.

Claims (21)

1. The method of cultivating human stem cells ex vivo, including cultivating a composition of human stem cells in a liquid culture medium under physiologically acceptable conditions, replacing the culture medium, characterized in that the replacement of the culture medium is carried out at a rate sufficient to ensure the division of human stem cells ex vivo, component 5 - 100% of daily replacement at a cell density of 1 x 10 ⁴ - 1 x 10 ⁷ per 1 ml of medium.  2. The method according to claim 1, characterized in that the stem cells of a human hematopoietic system are cultivated.  3. The method according to claim 1, characterized in that the stem cells of a human bone marrow are cultured.  4. The method according to p. 1, characterized in that they use a medium containing IL-3, GM-CSF, steel factor, Epo, IL-1α, IL-1β, G-CSF, the main fibroblast growth factor, IL-6 , IL-7, IL-8, IL-9, IL-10, IL-11, PDGF or EGF.  5. The method according to claim 1, characterized in that the composition of human hematopoietic stem cells is cultivated.  6. The method according to claim 1, characterized in that it further includes cultivating human stem cells in the presence of a gene transfer vector and receive stable genetically transformed human stem cells.  7. The method according to claim 6, characterized in that the human stem cells found in the hematopoietic system are cultivated.  8. The method according to claim 6, characterized in that the cultured human stem cells are hematopoietic cells.  9. The method according to claim 6, characterized in that the cultured human cells are peripheral blood mononuclear cells, bone marrow cells, fetal liver cells, embryonic stem cells, umbilical cord blood cells.  10. The method of claim 8, wherein said human hematopoietic stem cells produce stable, genetically transformed progenitor cells.  11. The method according to claim 6, characterized in that IL-3 and GM-CSF are added continuously to the specified environment.  12. The method according to claim 6, characterized in that the steel factor or IL-1α is added to the specified environment.  13. The method according to p. 12, characterized in that the steel factor and IL-1α are added to the specified environment.  14. The method according to claim 6, characterized in that the gene transfer vector is a retrovirus.  15. The method according to claim 6, characterized in that said human hematopoietic stem cells are cultured in the presence of a supernatant of a cell line packaging a retrovirus.  16. The method according to claim 1, characterized in that the human hematopoietic stem cells are cultured in the presence of transformed stromal cells attached to the matrix and capable of secreting at least one growth factor that directs the proliferation, differentiation or both of these hematopoietic cells moreover, the liquid culture medium is replaced daily and results in ex vivo division of human hematopoietic stem cells.  17. The method according to p. 16, characterized in that said human hematopoietic cells are bone marrow cells, and perfusion of the liquid culture medium and stromal cells support the division and production of human bone marrow stem cells, whereupon they are transfected.  18. Fibroblast CVI line of the African green monkey - producer of GM-CSF, which regulates the proliferation and / or differentiation of hematopoietic progenitor cells, transformed with the MTGM-CSFneo plasmid containing the DNA sequence encoding GM-CSF. 19. A method for separating hematopoietic neoplastic cells from normal cells, comprising applying a shear stress to the cell population that creates a shear stress sufficient to separate the neoplastic cells, characterized in that the population of hematopoietic cells is combined with stromal cells with limited mobility and contacting them, and shear stress create equal to or more than 1.0 dyne / cm ² . 20. The method according to claim 19, characterized in that the specified created shear stress is 1 to 1.2 dyne / cm ² . 21. The method according to claim 19, characterized in that said shear stress created is 1.2-1.5 dyne / cm ² .

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