RU2741769C2 - Biomedical cell product, method for its production and application - Google Patents

Abstract

FIELD: medicine; oncology; neurology.

SUBSTANCE: biomedical cell product for the treatment of oncological or neurological diseases comprises a line of autologous or immuno-compatible allogenic mesenchymal stromal stem cells (MSSC) with markers CD10+, CD13+, CD44+, CD90+ (Thy-1), CD105+, CD34−, CD45− and CD117− and mesenchymal stromal progenitor cells (MSPC) with markers CD10+, CD13+, CD44−, CD90+ (Thy-1), CD105+, CD34+, CD45+ and CD117+, recovered from native bone marrow or adipose tissue and cultured in vitro in a medium promoting mesenchymal differentiation (component A), non-manipulated autologous or allogenic mobilized mononuclear cells (MNC) recovered from peripheral blood leuconcentrate (PB LC), purified from blood plasma, erythrocytes and granulocytes (component B), and an auxiliary substance in the form of a sterile transfusion medium (component B), with the following ratio of components (vol % of the total amount of the BMLI): component A—1–19.5; component B—79.5–90; component B is rest. Group of inventions also relates to a method for obtaining ex tempore of said biomedical cell product and use thereof for treating oncological and neurological diseases.

EFFECT: group of inventions can be used in treating malignant neoplasms and neurological diseases.

10 cl, 9 tbl, 1 dwg

Description

The invention relates to the field of restorative medicine, oncology and neurology, and more specifically, to a biomedical cell product (BMCP) under the trade name "GemaComb", a method for producing this product and its applications. BMCP can be used for cytotoxic antitumor therapy of cancer and other malignant neoplasms, for restorative and rehabilitation treatment of cancer patients after conventional surgical antitumor treatment (surgical cytoreductive operations, stereotaxic radiosurgery, focused ultrasound, photodynamic therapy, thermal ablation of tumors, etc.), for the treatment of a wide range of severe complications arising from iatrogenic damage to human organs and tissues after drug chemotherapy and radiation therapy, for the prevention of progression / recurrence of cancer and malignant neoplasms and improving the quality of life of cancer patients, as well as for cell therapy and tissue engineering of organic nervous diseases and damage to the nervous tissue of the central nervous system.

Modern antitumor treatment of cancer and other malignant neoplasms includes a wide range of cytoreductive therapeutic effects (radical surgical removal of the tumor and adjacent lymph nodes) and a complex of cytostatic and cytotoxic effects (radiation therapy, drug chemotherapy, immunotherapy) on the neoplasm. This therapy is quite effective against tumors and systemically aggressive against organs and tissues of an oncological patient not involved in the pathological process. The successes of modern clinical oncology are obvious, since they significantly increased the five-year survival rate of cancer patients and extended the life expectancy of the population of highly developed countries of Europe and America by 15-20 years. However, in addition to successes and advances in modern clinical oncology, there are also serious problems associated with the correction of serious complications and side effects of standard antitumor cancer treatment and the prevention of recurrence and metastasis of malignant neoplasms, which require urgent solutions.

Modern high-tech surgical and microsurgical approaches to the treatment of tumors using intraoperative fluorescent dyes are capable of providing almost total removal of the tumor focus. However, often radical life-saving operations to remove tumors are crippling and become the main cause of disability and disability for cancer patients. It is known that the use of chemotherapeutic anticancer drugs in the treatment of cancer and malignant neoplasms is often accompanied by severe complications and side effects, which also lead to disability and often death of patients. Among such complications, reactions caused by the defeat of rapidly renewing (with a high rate of proliferation) cells of the hematopoietic and immune competent organs, primarily the bone marrow, prevail.

The toxic effect on hematopoiesis and immunopoiesis is the most frequent complication of modern chemotherapy and radiation therapy. By acting directly on the proliferating cells of the bone marrow, anticancer drugs are capable of inhibiting any hematopoietic germ. The strongest myelosuppressive effect is exerted by alkylating agents, anthracycline antibiotics, cytorabin, urea derivatives and taxanes. Predominantly the red sprout of hematopoiesis is affected by platinum preparations, methotrexate, chlorambucil, fludarabine. The genesis of the development of anemia manifests itself from a direct inhibitory effect (chlorambucil) on hematopoiesis to complete hemolysis (phlorambucil) of blood cells. In addition, antitumor agents are capable of damaging almost all normal structures and tissues of the patient's body with varying frequency, depending on the degree of toxicity of the drug. The frequency of different types of toxicity is not the same. The most common are gastrointestinal (up to 90% toxicity) and hematological (85-90%) toxicity, hepatonephrotropic toxicity is observed in 40% of cases, and cardiovascular toxicity in 45-50%. The defeat of the neuromuscular and respiratory systems is observed in 20-25% of cancer patients (Poddubnaya I.V., Orel N.F., 2015).

In recent years, in the practice of clinical oncology, drugs of a fundamentally new group of targeted action have begun to be used - targeted drugs that have a safety profile different from cytotoxic therapy. They inhibit hematopoiesis to a lesser extent, but cause specific side effects, such as skin lesions typical of EGF inhibitors, increased blood pressure and vascular changes (angiogenesis inhibitors), cardiotoxic effect (trastuzumab) (Poddubnaya I.V., Orel N.F. , 2015).

Anticancer radiation therapy for cancer patients also has serious complications and side effects. The defeat of organs and tissues in an oncological patient after radiation therapy consists in a pronounced local damage to the genome of cells, both of the organs and tissues themselves, which were directly irradiated by ionizing radiation, and in the general systemic radiation effect of radiation on the entire body of a cancer patient. As a result of the development of acute or chronic radiation sickness of varying severity in a cancer patient (Medvedev S.V., 2016), the entire symptom complex of radiation sickness requires immediate correction and active therapy. At the same time, the frequent occurrence of radiation necrosis and radiation edema of tissues (lungs, brain, etc.) is the most common cause of death in patients with tumors during radiation therapy.

Thus, as a rule, as a result of the successful complex standard antitumor high-tech treatment of cancer or other malignant neoplasms, an oncological patient in most cases is given the opportunity to temporarily stop the disease, achieve its remission and minimal residual manifestations, and save life for a certain time. Today, in oncology, the term has even been introduced - minimal residual disease (J.-F. Rossi, 2017; J.F. Rossi, 2018). However, for the temporary victory over the tumor, the oncological patient "pays" a very high price, in the form of systemic or micro-focal ischemic, hemorrhagic, dystrophic, inflammatory, degenerative lesions in the organs and endocrine glands, and also bears irreparable losses of the immune system, irreversible decrease in their own potential health and regeneration reserves. Radiation sickness, drug sickness, ischemic disease of organs and tissues, traumatic disease of tissues of internal organs are just a small set of real syndromic complications and undesirable side effects from classical antitumor therapy, which leads to irreversible changes and organic micro- and macrodefects in tissues and organs. After successful anticancer therapy of a tumor, an oncological patient develops an acute or chronic disease of damaged organs and tissues. Despite the fact that such an independent nosological unit as an iatrogenic "disease of damaged organs and tissues" after antitumor treatment does not exist in modern classifiers of diseases, every practicing doctor knows about its existence in an oncological clinic - oncologist-surgeon, radiation therapist, chemotherapist, doctor -oncogeneticist, immunologist, etc. This disease is masked under the flag of complications and side effects of standard cytoreductive, cytostatic and cytostatic anticancer therapy. The morphological manifestations of this disease in the affected organs are local acute and chronic inflammatory cicatricial-adhesive, cystic-cicatricial, degenerative-dystrophic adhesive processes. Sometimes its outcome is local and systemic inflammation and toxic or radiation (post-radiation) damage to organs and tissues of the body (dysmetabolic, electrolyte and hormonal disorders, instability of vital functions, necrotic changes, etc.) of an oncological patient. As a result of this disease, acute or chronic (cardiovascular, renal, hepatic, multiple organ, polyglandular, cerebrovascular, etc.) insufficiency of functions is formed, affected by the standard antitumor treatment of organs and tissues of the patient's body. Acute and chronic functional and morphological insufficiency of an organ affected by antitumor treatment can lead to complete incurability of an oncological patient, the impossibility of a repeated successful course of surgical treatment, radiation therapy or chemotherapy, and may become the main cause of death of this patient in the near future.

There are no drugs for the treatment of acute and chronic diseases of damaged organs and systems caused by the treatment of cancer. Symptomatic therapy or a palliative approach to the correction of complications after radiation therapy and chemotherapy, the fight against tissue edema or the consequences of radiation or post-radiation necrosis in the damaged area, rehabilitation and rehabilitation activities such as physiotherapy exercises and joint gymnastics are proposed. Physiotherapy is contraindicated in these patients, as it supposedly can enhance the neoplastic process. Cancer patients, as "lepers", are prohibited from sanatorium and rehabilitation treatment in places of general improvement of the population, although against the background of modern chemotherapy, the stimulating effects of impact on a malignant tumor as a result of rehabilitation and rehabilitation treatment are very doubtful. In this case, the toxic effect of radiation and chemotherapy on hematopoiesis (anemia, myelosuppression, bone marrow hypoplasia, neutropenia, cytostatic thrombocytopenia, etc., tissue inflammation, fungal infection, activation of a viral infection - herpes, CMV, Epstein-Bar, etc.) ) are obvious and absolutely real, and this action can only be partially corrected by erythrocyte mixtures, platelet concentrates, fresh frozen blood plasma, G-CSF (neupogen, granocid, neelastin, etc.), IL-11 (oprelvikin or neumega), the use of erythropoietin (EPO) (recormon, eprex, aranesp), iron preparations (Ferrum-Lek, maltofer, fenuls).

A similar situation has developed in clinical neurology. A huge number of patients who have suffered acute neurological diseases (brain and spinal cord injuries, hemorrhagic and ischemic stroke, demyelinating diseases and neuroinfections, neurosurgical operations to remove tumors of the brain and spinal cord, etc.) disorders that manifest limbs in the form of paresis and paralysis, impairments of various types of sensitivity, cognitive and intellectual-mnestic disorders, coordination disorders and other serious organic neurological syndromes and symptom complexes. At the same time, it has been proved that three years after exposure to an etiopathogenetic factor (infection, cerebrovascular accident, blow to the head, etc.), the agent that specifically led to the formation of an organic defect in the human brain and spinal cord (trauma , ischemia, hemorrhage, demyelination, tumor or inflammation, and more). In three years, the primary focus in the brain and (or) spinal cord of the victim will resolve with an outcome in a cyst or scar in the nervous tissue, as well as atrophy or degenerative-dystrophic process of the cells of the nervous tissue. Neoplastic transformation of damaged nerve tissue with the formation of tumor growth is also possible. Most often it is a combination of cicatricial adhesions or cystic-cicatricial, atrophic and adhesions. This is a natural result of the physiological sanogenesis of reparative and regenerative processes of homeostasis in the central nervous system (CNS) of a person. The severity of these organic lesions in the central nervous system depends on the volume and severity of damaged nervous tissue in the central nervous system, the effectiveness of the therapy of the underlying neurological disease, and the amount of neurorehabilitation and restorative treatment performed. Nevertheless, in this case, three years after the etiopathogenetic damaging effect, the anatomical and structural changes in the nervous tissue of the central nervous system are completed, and the entire complex of morphological and functional injuries that have occurred should be regarded as a disease of the damaged brain. Clinically, it can manifest itself both in the form of a chronic vegetative state (apallic syndrome, akinetic mutism, etc.) with a global loss of the main functions of the central nervous system, and in the form of minor or moderately expressed motor, sensory, coordinating and intellectual-mnestic and other dysfunctions Central nervous system. Unlike oncologists, neurologists and neurosurgeons classify these clinical manifestations within a year after trauma as residual effects of the transferred damage (trauma, ischemic infections, etc.), after a year - as the consequences of the transferred injury, and after 3 years - as long-term consequences. transferred damage to the nervous tissue of the brain and spinal cord. After 10 years or more, these changes are interpreted as residual organic changes in the nervous tissue in the central nervous system. There is no real treatment for this pathology in neurology, psychiatry, and neurosurgery yet. Treatment is symptomatic and syndromic.

To combat such conditions of acute and chronic organ damage in 2016, for the first time in the world, the American Food and Drug Administration (FDA USA) registered and licensed the innovative drug "HEMACORD ^® ", consisting of hematopoietic stem cells (HSC) obtained from umbilical cord blood. The drug is approved for clinical use to restore impaired hematopoiesis and correct complications after chemotherapy and radiation therapy of tumors (http://www.prnewswire.com/). It has been shown that due to the content of HSC and mesenchymal stromal stem cells (MSSC) in this preparation, it is effective in the treatment of toxic stomatitis, hepatotoxicity, cardiotoxicity, gastrointestinal disorders (toxic enteritis and enterocolitis), pulmonary toxicity and neurotoxicity.

In modern medicine, a new global biotechnological market for BMCP containing stem cells (SC) is actively being formed for oncology, neurology, restorative and regenerative medicine. The use of SC in BMCP allows to make up for insufficient functional activity of tissues and promotes the regeneration of damaged organs and tissues. The function of tissue renewal and restoration in vivo is performed mainly by tissue-specific SCs, which represent a pool of reserve undifferentiated precursors of cells of various types (Teplyashin A.S., 2005). Various types of tissue-specific SCs of an adult organism have been identified - HSCs with cell surface markers CD34 +, CD45 + (precursors of all blood cells), see RF patent No. RU 2283119); neuronal SC (NSC) with cell surface markers CD133 +, CD133- (precursors of nerve tissue cells), RF patent No. RU 2394593); MSSK with cell surface markers CD10 +, CD 13+, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- (cells capable of differentiating into tissue cells of mesenchymal origin), see RF patent No. RU 2252252, as well as SC of other germ layers.

The meaning of treatment with BMCP preparations containing SK is that the entire potential for a healthy and long life in the human body is in it from the very beginning. The human body contains special cells that constantly return us to normal - this is the SC. On the one hand, SC is the standard of the norm, from which normal cellular systems of any phenotype are able to form, and on the other hand, SC is the main controller and regulator of sanogenesis in the human body. Modern biotechnologies of medicine make it possible to take human SCs from the patient's or donor's body and use them in the form of BMCP for the treatment of diseases that were previously considered incurable (Teplyashin A.S., 2010; Bryukhovetskiy et al., 2014).

Currently, the main pathways and mechanisms of action by which SCs are able to restore damaged cells (PACs) in the tissues of the human body have been studied:

1. Pathway of functional and morphological substitution of POVCs by stem cells in the damaged area and directed differentiation of SCs into the POVC phenotype or POVA group.

2. The pathway of fusion of the cytogenetic biomaterial of the SC with the biomaterial of the POC for its regeneration and restoration.

3. The way of regulation and control of the pathways of intracellular signal transduction of POVA by molecularly targeted biologically active substances (microRNA, DNA, proteins) produced by SCs during intercellular interaction (S. Kuroda et al., 2013, Bryukhovetskiy A.S., 2014).

At this point in time, for a number of serious complications after surgical, chemotherapeutic and radiation treatment of cancer, BMCPs are practically the only way of possible treatment. In addition, the use of BMCPs containing SK increases the effectiveness of traditional methods of treatment, significantly facilitates the course of the disease and brings the moment of recovery closer (Baklaushev V.P., 2016).

It is necessary to distinguish both adult HSCs with markers CD34 +, CD45 +, HLA DR +, CD38-, Gp130- from hematopoietic progenitor cells (HCPs) with slightly different cell surface markers CD34 +, CD45-, HLA DR-, CD38 +, Gp130 +, and adult MSSCs with cell surface markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- from mesenchymal stromal progenitor cells (MSCP) with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1 ), CD105 +, CD34 +, CD45 + and CD117 +. In addition to differences in cell surface markers, HSC and MSSK differ from HSC and MSCB, respectively, in their fundamental properties and functions. Only adult SCs are capable of producing two SCs, or SCs and a CP (precursor cell) during division, while the CP is able to divide only into two SCs, which are not capable of producing SCs under normal conditions. Functional pluripotency is inherent only in the SC, and in the CP it is much lower than in the SC. PC is characterized by multipotency and a tendency to differentiate.

The very first and most effective experience of SC therapy, which made it possible to completely cure patients from blood cancer back in the 60s of the 20th century, was the experience of using the HSC preparation for transplanting bone marrow cells, that is, cells that are precursors of hematopoiesis. By transplanting HSCs obtained from the donor's bone marrow, it was possible to replace all hematopoietic cells (hematopoiesis) of the recipient and completely cure the patient suffering from acute myeloid leukemia (Blood Stem Cell Transplantation, edited by J. Reiffers, J.M. Goldman, J.O. Armitage, 1998). Therefore, the antitumor properties of HSCs in hematology oncology are the "gold standard" of SC therapy and a constant subject of study by scientists for over 70 years. Moreover, today it has become obvious that HSC, in addition to the formation of hematopoiesis, have an important regulatory and system-forming function in the body. In this regard, it is believed that the use of BMCPs made on the basis of GSK and MSSK is the most promising direction in modern medicine (Tupitsyn N.N. et al., 2014, DARPA report for 2011, D. Caplan, 2017). Already more than 100,000 patients around the world have received a licensed American drug "GEMACORD" containing cord blood HSC, and more than 520 cancer patients and more than 5700 neurological patients have received a combination of cellular systems and excipients, designated by us as BMCP "GemaComb". At the same time, 52 patients with severe spinal cord injury successfully received an experimental intramedullary (inside the spinal cord) injection of this drug into the damaged spinal cord or into a complete rupture of the spinal cord during operations on tissue engineering of the spinal cord. In the latter cases, the auxiliary substance in BMCP was the biodegradable heterogeneous matrix "SpheroGel" (patent RU 2249462, 2005), and the neuroprosthetic system was implanted into the spinal cord during neurosurgical operations on tissue engineering of the spinal cord.

GSK were first described by a Russian emigrant in the United States, Professor A.A. Maximov in 1908, as the progenitor cells of hematopoiesis. He showed that it is HSCs that are cells that form hematopoiesis, that is, the cells that underlie the entire process of hematopoiesis in humans and animals. But as a powerful therapeutic agent, these cells did not receive their well-deserved recognition at the beginning of the 20th century. For more than half a century they were forgotten and only in the mid-60s of the last century HSCs were successfully used to treat acute and chronic leukemias, lymphocytic leukemias and myelogenous leukemias in adults and children by bone marrow transplantation (Mentkevich G.L., Mayakova N.I. , 2010). The incredible successes in the treatment of blood cancer and even the complete cure of patients from this oncological ailment have led to the wide, but predominantly one-sided, mainly oncohematological, use of HSCs in the treatment of human diseases. Later, by the end of the 20th century, HSCs were widely used in most modern studies for the restorative treatment of impaired hematopoiesis in cancer patients after high-dose chemotherapy and radiation therapy of tumors. HSCs were used as a fundamental basis for bone marrow transplantation in the treatment of neoplastic formations, but no direct antitumor effect of HSCs on cancer cells has been proven (Bryukhovetskiy A.S., 2011).

HSCs with cell surface markers CD34 +, CD45 +, HLA DR-, CD38-, Gp130- in the human body are the most universal regulators of homeostasis, since they have the longest period of the cell life cycle (about 360 days) (Paltsev M.A. et al. , 2009). In the light of modern concepts of a systematic approach to management, it is obvious that in any biochemical process in the human and animal organism, the slowest phase is the governing one (Neimark J., 1985). In this regard, HSCs (CD34 +, CD45 +, HLA DR-, CD38-, Gp130-) in the human body have dominant control properties among all cellular systems of the body and their regulatory functions are systemic (Bryukhovetskiy A.S., 2010) for all normal cells the human body. Also, HSCs, depending on the organ needs of the tissues of the human body, are able, under the influence of signals from the microenvironment, to transform into both NSC and MSSK, which from the standpoint of the theory of "cell replacement" is extremely important for the restoration of damaged tissues.

Adult HSCs (CD34 +, CD45 +, HLA DR-, CD38-, Gp130-) have a unique ability of directed transfer to the zone of organ tissue damage, migrating to the gradient of inflammation concentration (patotropism or homing of the SC) (Baklaushev V.P., 2015, Bryukhovetskiy A. S., 2013). HSCs, entering the brain and spinal cord, are a powerful inducer of synapsogenesis in them or participate in the formation of new intercellular contacts in the tissues of solid organs (Bryukhovetskiy A.S., 2010, 2013).

It is known that when HSCs were transplanted into various organs (kidneys, brain, liver, etc.), their direct differentiation into specialized cells (cardiomyocytes, myocytes, or skin cells) of these organs was not observed, and the transplantation of precursor cells of hematopoiesis into the heart did not result in to the formation of neurons or intestinal secretory cells. Local differentiation in situ, as a rule, was controlled by “signals” from the microenvironment. The potential for transdifferentiation of HSCs into NSCs in vitro under the influence of certain perturbogens (retinoic acid) is well known (S. Kuroda et al., 2010). Long-term clinical observations also confirmed the absence of SC differentiation abnormalities in the graft. At the same time, HSCs have the function of purposeful migration to areas of damage (Chekhonin V.P. et al., 2005; Baklaushev V.P. et al., 2014) in the brain, as well as NSC and MSSK. In Russia, the technology of HSC transplantation for intracerebral administration in the treatment of a damaged brain was patented (patent RU 2216336, 2003), which was recommended by the authors for use in the treatment of Alzheimer's disease, Parkinson's disease and Creutzfeldt-Jakob disease. Similar properties of targeted migration to damage, homing, and patotropism are also characteristic of other tissue-specific SCs and their precursors, such as MSSK and NSC (Kaplan D., 2006; Snyder E. at al., 2006).

It has now been proven that transplanted leucoconcentrate (LA) cells of mobilized mononuclear cells (MNCs) formed clusters of HSCs and hematopoietic progenitor cells (HCPs) in the tissue of the damaged organ (Ono K. et al., 1999). In their own work, the authors of the present invention have shown that the multicellular cluster of mobilized MNCs, HSCs and HCPs of a person after introduction into the body (blood, cerebrospinal fluid, organ tissue) of rats behaved as system-forming in relation to the cells entering it and they migrated in a very organized and crowded predominantly (78%) to the affected organ, and then to the zone of maximum damage and were evenly distributed in this zone (Bryukhovetskiy A.S., 2013). E Snyder (2005) described a similar effect of NSC migration when injected into brain tissue from the right (intact) hemisphere to the left hemisphere, where a glial mouse brain tumor was simulated, in 36 days. The present inventors repeated this experiment in rats with C6 glioma by injecting LA mobilized MNCs containing HSC and MSSK and found that the migration of these cells towards the tumor hemisphere took 14 days. This phenomenon can be key not only in solving the problem of regeneration of damaged organs and tissues, but also in the development and creation of cell preparations based on HSCs. It is the cell cluster of transplanted hematopoietic cells that is of paramount importance for the transfer of these cells to the site of injury, as well as their targeted distribution in the area of damage to the pathological organ and adhesion of SCs to the affected cells, providing the optimal sanogenetic regulatory and restorative effects of SCs and their precursors on PACs.

Apparently, the role of HSC microenvironment cells in the restoration of disturbed hematopoiesis is decisive for the level of HSC functional activity both in the bone marrow niche and for HSCs transplanted into the blood (cerebrospinal fluid, tissue) as part of a cluster of bone marrow cells. In this regard, the authors of the present invention believe that in order to obtain the required functional (regulatory, antitumor, neuro-restorative, etc.) effect of HSCs in the human body, it is advisable to use them in the composition of a leuconcentrate containing the entire spectrum of cells of the microenvironment of the bone marrow niche. That is why, in the clinic of nervous diseases, the authors widely used this approach to inject HSCs into the body of a neurological and neuro-oncological patient with intraventricular or intrathecal transfusion. HSCs were necessarily injected as part of the mononuclear fraction of LA of mobilized peripheral blood (PC), since the effect of the introduction of a pure culture of HSCs was not observed either in the experiment or in the clinic. (Bryukhovetskiy A.S., 2010, 2011, 2013; Bryukhovetskiy A.S. et al., 2014, 2016). Interestingly, the isolated administration of MNC LK cells freed from HSCs (CD34 +, CD45 +, CD45-) does not provide the required neuro-restoration effects in the experiment in rats (Bryukhovetskiy A.S. et al., 2015).

As noted above, mobilized bone marrow MNCs are an important part of the bone marrow niche, in which HSCs and MSSCs are located, and microenvironmental cells, even in the PC, accompany the HSC and are of great importance in maintaining the activity and functionality of the HSC in the PC. HSC and MSSK and cells of their microenvironment, when administered intravenously, intraarterially or intrathecal to a person, move in biological fluids of the body in the form of an organized cell cluster. Apparently, cells of the natural microenvironment of HSC and MSSK provide the activity and systemic functionality of HSC and MSSK. In this vein, the use of HSC and MSSK in other nosological diseases is advisable to use in conjunction with the cells of their nearest microenvironment (Bryukhovetskiy A.S., 2013; Bryukhovetskiy A.S., Khotimchenko Yu.S., 2018). That is, the MNCs of mobilized blood are a prerequisite for the effectiveness of both our own and donor HSCs and MSSKs. It is not completely clear which cells of the microenvironment are decisive in the functional activity of HSC and MSSK; therefore, the authors of the present invention use the entire spectrum of the MNC cluster of mobilized blood in therapy.

HSCs implanted in the hemisphere opposite to the tumor or other pathological (inflammatory, ischemic, hemorrhage, etc.) focus, as well as those injected intravenously, demonstrate a common property of HSCs - targeted migration to the pathological focus and restoration of disturbed interstitial homeostasis (Bryukhovetsky A. S., 2011; Bryukhovetskiy I.S. et al., 2015). This property of HSC is used for superselective delivery of immunoliposomal constructs containing antitumor chemotherapy drugs or other molecules of cytotoxic or cytostatic action (patent RU 2336901, 2008). In addition, superselective delivery of radiopaque substances, radioisotope drugs, signaling proteins and nanostructures that increase the sensitivity of tumor cells (TC) to traditional methods of therapy is possible (Bryukhovetskiy A.S., 2011). The strategy of using HSC as a transport system for targeted delivery of biological information for local modulation of apoptosis processes seems to be promising, which requires further research in this direction (Bryukhovetskiy I.S. et al., 2014, 2016). Similar properties of SCs, characteristic of HSCs, are described for ISSCs (Caplan D., 2013, 2014; M. Choop et al., 2015, 2016).

The studies of the authors of the present invention have shown a fairly high efficiency of mobilization of HSCs (CD34 +, CD45 +) and MSSK (CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117-) in PC in patients with non-oncological diseases ( up to 95.1%). It is well known that under conditions of calm, undamaged hematopoiesis, HSCs and MSSKs necessarily circulate in the human PC, however, their concentration is so negligible (less than 0.01%) that makes their detailed study or, moreover, their use for transplantation purposes practically impossible. The release of HSC and MSSK from the central organs of hematopoiesis (bone marrow) is observed after trauma to hematopoiesis (most often, as a result of chemotherapy), as well as under the influence of colony stimulating factors (CSF). In the clinic, the most widespread are granulocytic CSF (G-CSF) and granulocyte-macrophage CSF. Under the influence of these factors, the concentration of HSC and CP in the blood increases 100-1000 times, which makes it possible to collect these cells and use them for transplantation in the clinic. Collection of HSCs in these cases was mainly carried out for the subsequent restoration of impaired hematopoiesis after high-dose chemotherapy. In cancer patients, due to previous chemotherapy, the bone marrow is in a certain sense "depleted" at the time of mobilization, and effective mobilization of SC (more than 20 cells in μl of blood) was not achieved in all cases. As a rule, it was possible to gain the number of cells required for transplantation (2⋅10 ⁶ / kg of the patient's weight) only after 2-3 sessions of leukocytopheresis.

It is known that the use of isolated HSCs (CD34 +, CD133 +, CD45 +) to restore disturbed hematopoiesis after high-dose chemotherapy or radiation therapy during bone marrow transplantation in cancer patients turned out to be absolutely ineffective, and all patients who received only isolated HSCs (CD34 +, CD133 +, CD45 +), died, since these cells did not take root in the recipient's bone marrow, that is, a pure culture of HSC and MSSK cells (CD10-, CD 13+, CD44-, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117-) was not able to provide restoration of damaged hematopoiesis (Blood Stem Cell Transplantation, edited by J. Reiffers, JM Goldman, JO Armitage, 1998). These data have been confirmed in the work of domestic oncotransfusiologists (Mkheidze D.M. et al., 2012) and bone marrow transplant specialists (Mentkevich G.L. et al., 2013). That is why the use of only one purified HSC (CD34 +, CD133 +, CD45 +) to restore disturbed hematopoiesis is considered methodically erroneous and incorrect. At the same time, if the total amount of HSCs in the transplanted material of a leukopheresis product or bone marrow is less than 1%, then the risk of an unfavorable outcome of the restoration of impaired hematopoiesis after bone marrow transplantation doubles (L.Yu. Grivtsova, 2017).

Despite the fact that HSC transplantation and bone marrow transplantation have been carried out in Russia for more than 70 years, there are no special domestic preparations containing HSC, and HSC transplantation and selection of bone marrow donors are carried out spontaneously, only as "bone marrow transplantation" and only on the basis of complex histocompatibility HLA types I and II and the number of HSCs. There are also no official cellular preparations for restoring damaged hematopoiesis after chemotherapy and radiation therapy. With the suppression of the myeloid lineage during these therapies (mainly with irradiation of flat bones - the sternum and pelvis), neutropenia is formed quickly (7-12 days) or delayed (3-6 weeks), which is dangerous for the development of infectious complications, fungal lesions, skin lesions (mucoside, wounds, cracks), concomitant immunodeficiency and cytomegalovirus infection join. Antibiotics are added to the treatment for the treatment of interstitial infection and G-CSF (Neupogen, Granocid, Neupomax, Granovesil, etc.). Thrombocytopenia is dangerous by the development of hemorrhagic diathesis, therefore thrombocytopenic concentrate is used. Thus, the issue of new cell products that can be used to treat complications and side effects from tumor therapy is extremely relevant for modern domestic and world oncology.

In July 2016, Russia adopted Federal Law No. 180 FZ "On Biomedical Cell Products", which at the state level regulates the development and creation of BMCPs, preclinical studies and limited clinical trials of BMCPs, circulation of BMCPs and their widespread use in the clinic in humans. Therefore, the relevance of creating new cellular products is obvious, since until now in Russia they talked mainly about cellular technologies and SCs, and not about BMCP. By a biomedical cell product, this law means a complex consisting of a cell line (cell lines) and excipients, or a cell line (cell lines) and excipients in combination with state-registered medicinal products for medical use and / or medical devices.

As the closest analogue, i.e. The prototype of the present invention adopted a preparation of autologous HSCs for transplantation with lesions of the nervous system and a method for producing this preparation (patent RU 2283119, 2006). This known preparation is autologous cells obtained from a patient's PC, enriched with HSCs containing the CD34 antigen, at a final concentration of (40-100) × 10 ⁶ cells / ml.

A unique feature in the process of preparing this well-known cell preparation was that, unlike mobilized HSCs and MSCs for the treatment of cancer patients, G-CSF was administered for 6-7 days (the classic version of HSC stimulation), which made it possible to obtain mature HSCs (CD34, CD133 +, CD45 +), and stimulation with G-CSF was used only for 4 days and as a result of taking on the 5th day, stimulation was obtained from the PC mainly HCP (CD34 +, CD133, CD45-). This drug is still successfully used to date for the treatment of extremely severe neurological patients with traumatic diseases of the brain and spinal cord. However, when compiling this known drug and its use, the role of MSSK in it was absolutely not taken into account, and now a higher efficiency of BMCP of this type is required.

The objective of the present invention is to create a new BMCP using SCs mainly for the needs of modern oncology and neurology. The technical results achieved with the use of BMCP according to the present invention are effective restoration of the myeloid lineage and the entire hematopoiesis during the correction of complications and restoration of cellular damage after radiation and chemotherapy, as well as effective healing of organic damage to the human brain and spinal cord.

According to the first aspect of the present invention, the solution to this problem is achieved by the fact that the proposed BMCP for the treatment and prevention of cancer and neurological diseases, containing a line of autologous or immunocompatible allogeneic (from an alloreactive, and preferably from a haploidentical donor) mesenchymal stromal stem cells (MSSC) with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and mesenchymal stromal progenitor cells (MSCP) with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + isolated from native bone marrow or adipose tissue and cultured in vitro in an environment promoting mesenchymal differentiation (component A), unmanipulated or poorly manipulated autologous or allogeneic mobilized mononuclear cells (MNC) isolated from peripheral blood leuconcentrate (PC PC) purified from blood plasma, erythrocytes and granulocytes (component B), and an auxiliary substance in vi de biologically stable and sterile transfusion medium (component B) with the following ratio of components (vol. % of the total number of BMKP):

component A - 1-19.5;

component B - 79.5-90;

component B - the rest.

Mainly, component B contains αβ T cells, γδ T cells, NK and NKT cells, B1 and B2 cells, hematopoietic stem cells (HSC) with markers CD34 +, CD45 +, HLA DR +, hematopoietic progenitor cells (HSC ) with markers CD34 +, CD45-, HLA DR-, MSSK with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD 117- and MSCC with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + with the following ratio of components (vol.% Of the total amount of component B):

αβ T cells - 55-60;

γδ T cells - 10-15;

NK and NKT cells - 15-20;

B1 and B2 cells - 10-15;

GSK and GKP - 1.5-2.5;

ISSK and ISKP - 1.5-2.5.

In one embodiment of the present invention, component B is partially purified from αβ T cells at the following ratio of components (vol.% Of the total amount of component B):

αβ T cells - 30-55;

γδ T cells - 15-20;

NK and NKT cells, 17-30;

B1 and B2 cells - 10-15;

GSK and GKP - 1.5-2.5;

ISSK and ISKP - 1.5-2.5.

As an auxiliary substance, 0.9% saline NaCl solution with granulocyte colony-stimulating factor (G-CSF) in a volume of 5-100 vol. %. from the volume of a physiological solution of NaCl or a biodegradable polymer matrix with a granulocyte colony-stimulating factor (G-CSF) transfused in it in a volume of 5-20 vol. % of the volume of the matrix.

Predominantly, allogeneic MNCs are immunocompatible with respect to blood group, Rh factor, and blood immunophenotype.

According to the second aspect of the present invention, the solution to the above problem is achieved by the fact that there is provided a method for ex tempore preparation of a biomedical cell product (BMCP) proposed by the present invention, comprising:

obtaining a line of autologous or immunocompatible allogeneic mesenchymal stromal stem cells (MSSC) with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and mesenchymal stromal progenitor cells (MSSC) with markers CD10 , CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + by isolating these cells from native bone marrow or adipose tissue and further cultivation in vitro in an environment promoting mesenchymal differentiation (component A),

obtaining unmanipulated or little-manipulated autologous or allogeneic mobilized mononuclear cells (MNC) by their isolation from peripheral blood leuconcentrate (PC PC) with subsequent purification from blood plasma, erythrocytes and granulocytes (component B) and

ex tempore mixing of components A, B and an auxiliary substance in the form of a biologically stable and sterile transfusion medium (component C).

If necessary, the obtained components A and B are cryopreserved and defrost before mixing ex tempore with component C.

According to further aspects of the present invention, there are provided the uses of the BMCP of the present invention for the treatment and prevention of cancer and other malignant neoplasms, as well as for the treatment of neurological diseases.

In modern world immunology, regenerative medicine and transfusiology, unmanipulated or poorly manipulated cells are understood to mean human and animal cell systems that, as a result of isolation, purification, processing, standardization, certification, cryopreservation and storage, are not subjected to genome modification processes, cultivation or prolonged (more than 12 hours ) incubation of the obtained cells with chemicals or drugs, as well as cells, as a result of manipulations with which the native (natural) genetic and postgenomic characteristics of the transcriptome, proteome, metabolome and secretome of these cells remain unchanged.

FIG. 1 shows a diagram of the immunophenotypic characteristics of HSC cell surface markers (CD34 +, CD 133+) in the norm (donors), in neuro-oncological patients and patients with amyotrophic lateral sclerosis (ALS).

Т-клеток (системы врожденного иммунитета), способных уничтожать опухолевые клетки пациента. В-третьих, предложенный БМКП способен оказывать мощное иммунное эффекторное воздействие на ОК, мобилизуя свои противоопухолевые эффекторные клеточные системы врожденного иммунитета (NK-клетки, NKT-клетки,

Т-клетки, В1-клетки), находящиеся в ЛК МНК. В-четвертых, при правильном расчете количества МССК и ГСК в предложенном БМКП можно добиться реального воздействия на пролиферативные, репродуктивные и миграционные свойства раковых стволовых клеток (РСК), что сегодня не способен обеспечить не один фармацевтический таргетный противоопухолевый препарат.В итоговом отчете о прикладных научных исследованиях по теме «Разработка биомедицинского клеточного препарата гемопоэтических стволовых клеток для персонифицированной протеом-основанной терапии опухолей головного мозга», выполненного в рамках ФЦП "Исследования и разработки по приоритетным направлениям развития научно-технологического комплекса на 2014-2020 годы" (шифр заявки 2014-14-576-0057-078) авторами настоящего изобретения было показано, что при соотношении количества здоровых ГСК или МССК к ОК равном 3 к 1 in vitro и in vivo функциональная активность РСК блокируется путем горизонтального и вертикального переноса регуляторных белков из СК в РСК.According to the present invention, an alternative to the American drug "HEMACORD" is proposed, a new BMCP with the trade name "GemaComb". The proposed BMCP, as well as "HEMACORD", contains HSC and MSSK and is aimed at restoration (restoration) of cells of damaged tissue of the body of a cancer patient, obtained as a result of various types of toxicity after conventional and targeted anticancer therapy, and also has advantages that are not available in the American product. Firstly, MSSK and HSC used in the proposed BMCP are in the usual tissue-specific microenvironment of the cells of the bone marrow niche, which significantly increases their efficiency and biological activity. Secondly, the work of Japanese researchers has shown (K. Ono et al., 2001) that the LA cells of mobilized bone marrow MNCs, when infused into the recipient's blood or into the tissue of the damaged organ, migrate to the damaged areas within the bone marrow cluster, which allows increase the survival rate of SCs many times and achieve a more efficient homing of these cells to the damaged areas. In the case of the correct molecular genetic selection of component B, the antitumor activity of BMCP increases many times due to the alloreactivity of these cellular systems due to the presence of NK-, NKT-cells and

T cells (innate immune systems) capable of destroying the patient's tumor cells. Thirdly, the proposed BMCP is capable of exerting a powerful immune effector effect on TC, mobilizing its antitumor effector cellular systems of innate immunity (NK cells, NKT cells,

T cells, B1 cells), which are in the LC MNC. Fourth, with the correct calculation of the amount of MSSK and HSC in the proposed BMCP, it is possible to achieve a real effect on the proliferative, reproductive and migration properties of cancer stem cells (RSC), which today is not able to provide more than one pharmaceutical targeted anticancer drug. research on the topic "Development of a biomedical cell preparation of hematopoietic stem cells for personalized proteome-based therapy of brain tumors", carried out within the framework of the Federal Target Program "Research and Development in Priority Areas of Development of the Scientific and Technological Complex for 2014-2020" (application code 2014-14 -576-0057-078), the authors of the present invention have shown that when the ratio of the number of healthy HSCs or MSSKs to TCs is 3 to 1 in vitro and in vivo, the functional activity of CSCs is blocked by horizontal and vertical transfer of regulatory proteins from SCs to CSCs.

The authors of the present invention in the process of developing BMCP according to the present invention conducted research and modifications of the above known preparation adopted as a prototype (patent RU 2283119, 2006). Until 2008, this well-known preparation used only autologous biomaterial (obtained from the patients themselves), and since 2008, they began to use allogeneic donor material and for 3-7 days cultivate a standard leucoconcentrate with a growth medium that prevents differentiation, but allows HSC to grow. up to 2% or more, resulting in an increase in product efficiency. Subsequently, they began to enrich the LA MNCs of the bone marrow with cell systems from the MSSK line of allogeneic healthy donors, both for the purpose of neuro-restoration and for the purpose of restoring damaged bone marrow and correcting the consequences of radiation and chemotherapy in cancer patients. The addition of the allogeneic MSSK cell line and the cultivation of LA MNCs for 3-7 days ensured a high purification of the preparation from the remaining erythrocytes and granulocytes and increased the efficiency of the MSSK product for use in oncological and neurological practice.

Thus, the biotechnological platform for creating BMCP according to the present invention under the trade name "GemaComb" or in other words, the basic cellular matrix of the product according to the present invention is based on the widely used by the authors of the invention (Bryukhovetskiy A.S. et al., 2010-2016; Dolgopolov IS et al., 2010) from 2002 to 2018 from an autologous (own) or allogeneic (donor) immunocompatible (by blood group, Rh factor, immunophenotype) cell suspension of MNC mobilized PK, containing a small (0.5-2 %) the number of HSCs (CD34 + CD45 +) and HSCs (CD34 + CD45-), as well as the minimum amount (1-1.5%) of MSCs (with cell markers CD10-, CD 13+, CD44-, CD90 + (Thy-1 ), CD105 +, CD34-, CD45- and CD117-), which were enriched with donor MSSK obtained from the cell line under conditions of production of the GMP standard. The MSSK cell line had cell systems with cell surface markers CD 10+, CD 13+, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and MSCs (CD10 +, CD 13+, CD44-, CD90 + (Thy-1), CD105 +. CD34 +, CD45 + and CD117 +). The source of obtaining allogeneic MCSCs was from 2-5 ml of bone marrow exfusate from the patient himself or from a healthy donor, or 5-10 ml of the LC MNC of the bone marrow of the patient himself or a healthy donor. In some cases, the MSSK line was obtained from the adipose tissue of a patient or donor by surgical excision of it on the anterior abdominal wall or obtained by the standard liposuction procedure.

The amount of HSCs used in this proposed BMCP is 1-2% of the mass of the total LK MNC PC and this amount is quite sufficient for those regulatory and systemic functions that these cells must perform during infusion to a patient. However, the amount of MCS in a given LK MNC PC is no more than 1%, and this amount of SCs required for neuro-restoration purposes of reconstructing a damaged brain or blocking proliferative, migratory reproductive functions is clearly insufficient. Therefore, in the proposed BMCP, mobilized cells of LA MNC PK (component B) are enriched in adult MSSCs with cell surface markers CD10 +, CD 13+, CD44 +, CD90 + (Thy-1), CD105, CD34-, CD45- and CD117- and MSCs with markers CD10 + , CD 13+, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + from a cell line derived from bone marrow or adipose tissue (component A).

The complete structure of mobilized MNCs, precursors of mouse and human PCs, is represented by natural killer cells (NK cells, NKT cells), T cells (T lymphocytes: γδ T cells, αβ T cells), HSC (CD34 +, CD133 +, CD 45 +, HLA DR +) and HCP (CD34 +, CD133 +, CD45-, HLA DR-), MSSK (CD10 +, CD 13+, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117-) and MSS (CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 +), B cells (B lymphocytes: B1 and B2 cells). Mobilized PBMCs are a very heterogeneous structure of hematopoietic, immune, and blood stem cells at various stages of differentiation. The use of granulocyte colony-stimulating factor (G-CSF) for the mobilization of these cells within 4 days ensured the constancy of the composition of the multicellular matrix of immune, hematopoietic and progenitor cells.

Considering that αβ T-cells (T-helpers, T-suppressors, Tregs), the content of which in the above-described initial preparation is 50-60%, are the main source of the graft-versus-host reaction, the use of this drug has significant limitations in a large the number of cancer patients and certain difficulties with intravenous administration in neurological patients.

Т-лимфоциты, ГСК и ГКП, МССК) здорового донора в количестве 45-50% клеточной массы. Таким образом, ЛК, истощенный αβ Т-клетками, будет содержать γδ Т-клетки (лимфоциты) в количестве 20-30%, αβ Т-клетки 5-10%, NК-клетки и NKT-клетки - 34-40%, В1-клетки -до 10% от общего количества клеток, ГСК и ГКП - до 4-5%, МССК - до 4-5%.To solve this problem, the present invention provides for the partial purification of component B from αβ T cells, when depletion or depletion of αβ T cells is performed using the CliniMACS® plus apparatus clinically used for immunomagnetic cell selection using standard methods of immunoseparation with CliniMACS TCR alpha / beta Kit antibodies. However, for the treatment of neurological patients, such depletion of αβ T cells is not necessary. In the processed and manipulated LA of mobilized MNCs, all the main effectors of innate immunity remained (NK cells and NKT cells, B1 cells,

T-lymphocytes, HSC and HKP, MSSK) of a healthy donor in the amount of 45-50% of the cell mass. Thus, LA depleted by αβ T cells will contain γδ T cells (lymphocytes) in an amount of 20-30%, αβ T cells 5-10%, NK cells and NKT cells - 34-40%, B1 - cells - up to 10% of the total number of cells, HSC and HCP - up to 4-5%, MSSK - up to 4-5%.

Thus, as a result of the standard procedure of leukocytopheresis, the LA of mobilized PB MNCs is obtained, and after its enrichment with various types of SC, a universal biotechnological cellular platform for a whole line of various anticancer drugs was obtained. The addition of HSC or MSSK to this cell culture platform significantly increases the potential of this drug against cancer SC (CSC). Enrichment of this natural mononuclear cell platform with a line of allogeneic SCs (HSC, MSSK, NSC) significantly increases its antitumor properties, on the one hand, and increases the regenerative potential, on the other hand.

The number of required cultured donor MSSK is determined by simple calculations. The amount of OC in the patient's body is calculated using various methods (for example, the Kshivts method). The content of RAC is 1-2% of the amount of TC in the patient's body. Multiplying the number of CSCs by a factor of 3 (the ratio that blocks the function of CSCs is 3 HSCs or MSCs per 1 CSCs), the optimal amount of SCs required to block the functions of CSCs in a particular patient is obtained. The resulting BMCP according to the present invention was administered to various oncological and neurological patients intravenously or intrathecally, taking into account the compatibility of the blood group and Rh factor of the donor and recipient. Although, with good purification of the LA from erythrocytes and granulocytes, these indicators are not required.

In some cases, the LA of mobilized MNC cells were cultured for 3-7 days in a medium containing interleukin-2 (IL-2). The cultivated LA was evaluated after cultivation according to its cellular composition to determine the percentage and quantitative content of basic cellular elements in it (HSC, HCP, MSSK, NK cells and NKT cells, γδ T lymphocytes and B1 and B2 cells). After 3-5 days of cultivation, these MNC cells could be used in a patient without taking into account group and Rh factor compatibility, since there were no more erythrocytes and granulocytes in this cell suspension.

The combination of PB MNCs containing HCPs and MSSKs with adult hematopoietic SCs (HSCs) and / or mesenchymal SCs (MSSKs) ensures the formation of optimal cellular symbiosis within a cluster of hematopoietic and immune cells, in which the functional and regulatory properties of HCPs and HSCs, as well as MSCs and MSSKs are maximally active and quite physiological. The formation of a mononuclear therapeutic cluster of hematopoietic cell systems in BMCP cultivated for 3-7 days in a growth medium, which prevents differentiation, makes it possible to create a fundamentally new biologically active composition of cellular systems capable of the fastest and optimal restoration of disturbed hematopoiesis, restoration of damage in organs and tissues of oncological the patient after surgical resection of the tumor, radiation therapy and (or) chemotherapy, and also allows you to prevent tumor recurrence and further spread (metastasis) of the tumor in the body of the cancer patient. But most importantly, this allows not to lose, but on the contrary, to increase the number of HSCs in the LC of mobilized MNCs, in which the amount of HSCs is decisive for the successful transplantation of bone marrow cells. The use of short-term (3-5 days) cultivation of LA MNC PC in a growth medium that prevents differentiation and contributes to an increase in the number of HSCs, makes it possible to improve the quality of this drug and increase the potential for its use for HSC transplantation and restoration of the damaged hematopoiesis process in cancer patients after radiation and chemotherapy. ...

The creation of a system-forming and biologically active mononuclear multicellular cluster of mobilized hematopoietic and immune cells containing precursors of hematopoiesis and donor HSCs and MSSKs is of great importance for the reconstructive-restorative effect of the proposed BMCP. Historically, the role of HKP in bone marrow transplantation was practically not taken into account in the LC of mobilized MNCs, since for this type of therapy it is the amount of HSCs that is decisive for the restoration of impaired hematopoiesis. But it is the large number of HKPs in the LA of mobilized MNCs that potentiate and determine the pronounced clinical effects of an insignificant amount of HSCs, and for other therapy tasks is also the most important therapeutic component. For more than 14 years, the authors of the present invention have widely used the matrix of the proposed BMCP from autologous or haploidentical HCPs and MSCs in MNCs in the treatment of various nervous diseases in humans as an independent cell preparation and have extensive clinical experience (more than 15,000 transfusions) in the use of drugs containing HCPs in the treatment 5807 patients. The drug was used in the clinic of nervous diseases for various neurological and oncological patients intrathecally (injection into the cerebrospinal fluid), intraventricularly (infusion into the ventricular system of the brain), intravenously and intraarterially. The authors of the present invention believe that the most attractive for the impact on the pathological focus in the human body are the autologous HKP and HSC (Bryukhovetskiy A.S., 2005-2016, Bryukhovetskiy AS et al., 2006-2016), since against the background of their long-term systemic use, a convincing neuro-restorative (neuro-restorative), morphological and clinical effect was noted (57-61.2% of cases), and there were practically no life-threatening situations and complications in patients. These lines of GKP and ISKP have already received their own vector of differentiation, and the technology for their production does not present any great difficulties (prototype under patent RU 2283119). All the complications that were observed during the use of the proposed BMCP were quite understandable and were easily relieved symptomatically, without leading to disturbances of vital functions or serious consequences.

Experiments and simple mathematical calculations were carried out to determine the necessary and sufficient number of GSK and MSSK in the proposed BMKP. The experiment on modeling the intercellular interaction of HSCs with pathological cells is most clearly demonstrated in model cancer systems. The results of studies on this issue were published in the journal "Genes and Cells", volume XI, No. 3 in 2016 in the original article (Milkina E.V. et al., 2016). This article shows that any interaction between living objects or cellular systems is the transfer of information, which is fully consistent with the literature data (Paltsev M.A. et al., 2003). It has been shown, for example, that the migration activity of HSCs in relation to glioblastoma cells exceeds their "mobility" relative to TC of other types, which indicates a high information potential of glioblastoma cells. The ability of glioblastoma to recruit cells of various types described in the literature (de Fonseca A.S., Badie B., 2013; Rishardson, 2015) confirms this observation. As is known, HSCs migrate to the damaged area along the concentration gradient of chemoattractants. The main role in this process belongs to stromal cell factor 1α (SDF-1α). In addition, another 80 ligands produced by damaged cells and tissues have been described. In this case, the main inducer of the synthesis of these molecules are highly active molecules of the family of HIF-factors induced by hypoxia (Bryukhovetsky I.S., Bryukhovetsky A.S., Mishchenko P.V. et al., 20013). Hypoxia, one of the key criteria of glioblastoma multiforme (MGB), triggers the mechanisms of clonal selection, which sharply increases the invasive potential of the tumor, as a result of which the number of exosomes secreted by OC and transporting a wide range of molecules that also contribute to the migration of SC to the tumor focus increases (Hu J., Sun T., Wang H. et al., 2016; King HW, Michael MZ., Gleadolle JM, 2012; Iwadate Y., Matsutani T., Hirono S. et al., 2016). In the processes of invasive growth and metastasis of malignant tumors, a special role belongs to the epithelial-mesenchymal transition - a complex process of epithelial cells changing their phenotype to mesenchymal, which is accompanied by profound changes in the cellular proteome (Iwadate Y., 2016; Kubelt Hatterman K., Sebens et al., 2015 ). As shown experimentally, stimulation of human glioblastoma cells with transforming growth factor 1β changes the molecular phenotype of human glioblastoma cells U87 (Bryukhovetskiy I., Shevchenko V.E., 2016). The production of TGF-1β increases under hypoxic conditions, while the maximum concentration of TGF-1β was observed around necrotic areas in the glioblastoma tissue (Iwadate Y., Matsutani T., Hirono S. et al., 2016), where normal SCs that migrated into the tumor accumulate, producing exosomes (Bryukhovetskiy I., Bryukhovetskiy A., Chotimchenko Y. et al., 2016).

Research by specialists in the immunology of hematopoiesis at the FSBI "Russian Cancer Research Center. N.N. Blokhin of the Ministry of Health of the Russian Federation (Grivtsova L.Yu., Tupitsyn N.N., 2016) showed that in a deep study of the cell surface markers of autologous HSCs and adult HSCs, which were used to treat various patients, the immunophenotype of HSC markers is heterogeneous and differs greatly in healthy donors. , adult oncological patients and children suffering from various oncological diseases, and differs significantly in patients with degenerative and atrophic diseases of the nervous system. Despite the fact that the clinical analysis of blood did not show quantitative disturbances and formal changes in the blood formula and disturbances in the morphology of the structural elements of the blood in these patients, their hematopoiesis (hematopoiesis) at different stages of the disease differs greatly and seriously suffers at the level of regulatory cellular systems of the congenital and acquired immunity.

As seen from the diagram in FIG. 1, in patients with amyotrophic lateral sclerosis (ALS), the CD38 marker is practically absent on the surface of autologous HSCs, and the indicators of CD117 and CD71 are sharply reduced, and in patients with oncological diseases on the surface of HSCs, a significant suppression of the CD33 marker and markers CD71 and CD90 (Thy -one). The total number of leukocytes in the LK of the bone marrow MNC is (10-20) ⋅10 ⁹ cells, that is, of which HSC and HCP are at least 1%, that is, (1-2) ⋅10 ⁶ cells. Therefore, for the restoration of hematopoiesis of these cells, it may be and will be quite enough, but for the suppression of the regulatory properties of OC and CSC by healthy HSCs in the amount of 0.5-1%, they will obviously not be enough. Considering that, for example, the amount of CSCs (gliomaspheres) of glioblastoma multiforme is from 6-15%, then, depending on the tumor volume, the amount of MSSK should be in the range (100-300) × 10 ⁶ . And this is possible only with the enrichment of LA MNCs by cells of the MSSK cell line. Also, the amount of HSC and HSC in the preparation is quite sufficient to enhance the clinical effect of the substitution and regenerative effect of autologous HSC and HSC on the affected organ tissue or massive exposure to biologically active substances and regulatory substances produced by HSC. However, in case of massive damage to the nervous tissue, the amount of MSSK should be at least (30-60) × 10 ⁶ . Also, this amount of HSC and HKP is quite effective for the occurrence of an adequate clinical effect both in neuroregeneration and in restoring homeostasis and restoration of damage in organs and tissues. However, the number of MCSs required to block RACs and their predecessors is clearly insufficient.

For the last 10 years, the research group, which included the authors of the present invention, has been engaged in serious fundamental scientific research in the field of intercellular interaction of SCs and various types of OC and CSCs in vitro and in vivo (Bryukhovetsky A.S., 2003-2016; Bryukhovetsky I.S. et al., 2010-2016; Bryukhovetskiy AS et al., 2013-2016; Bryukhovetskiy IS et al., 2010-2016). The main scientific fact of these studies was convincing experimental evidence of the biological effect of the horizontal information transfer of micro- and nanoparticles of the cytoplasm of the SC in the OA and CSC and vice versa. It was proved that horizontal information exchange between these cellular systems is the main tool for the regulation of HSCs and is carried out by secretion of HSCs of multivesicular vesicles of various diameters (from 10 to 1000 nm), which have a powerful antitumor (antiproliferative, anti-migratory, antireproductive, etc.), and also pronounced neuro-regenerative and regenerative potentials. The task was set to trace the movements of proteins and RNA between HSC, OA, and RSK. Obviously, proteins and RNA, after being absorbed by the target cell, can be determined only by labeling them with special fluorescent labels, which allow tracking such movements by means of fluorescence microscopy. For this, the cytoplasm of OK, RSC and HSC were labeled with fluorescent dyes. As a result of these studies, it was shown for the first time in the world that when the ratio of three HSC cells to one OC or one CSC (3: 1) in the pathological focus, the main functions (reproduction, mitosis, migration, etc.) of HSCs are completely blocked. It is the establishment of the fact of blocking the functioning and reproduction of TC and CSC due to the regulatory inhibitory effect of regulatory proteins and RNA of these HSCs in a tumor (Bryukhovetskiy I.S. et al., 2016) that became the main achievement of these studies. However, in the interaction of HSCs with POCs (non-neoplastic) at a ratio of 2 HSCs to 1 POCs, opposite tumor sanogenetic processes of POC regeneration and restoration are triggered (Bryukhovetskiy I.S. et al., 2016).

Therefore, the use of HSCs (CD34 +, CD45 +) and HSCs (CD34 +, CD45-), contained only in the LAs of mobilized MNCs of the patient's PC, is clearly sufficient not only for the regeneration and restoration of POC after radiation or chemotherapy (HSC: POC = 1: 2), but and even more so to provide an antitumor effect (GSK: RSK or OK = 3: 1).

On the basis of the above studies, according to the present invention, a scientifically grounded, balanced, therapeutic BMCP with increased antitumor and regenerative properties was created, which is a combination of MNC mobilized PK (component B), enriched in cell systems of the cultured MSSK line with cell surface markers CD10 +, CD 13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and MSCPs with cell surface markers CD10 +, CD 13+, CD44-, CD90 + (Thy-1), CD105 +. CD34 +, CD45 + and CD117 + (component A) and placed in an auxiliary medium (component B) in the form of 0.9% saline solution of sodium chloride or a biodegradable polymer matrix, which can be used as a matrix "Spherogel" described in patent RU 2249462, 2005 year

Cultivated for 2-6 days under special conditions, LA of mobilized MNCs PC becomes highly effective for intravenous use in cancer patients to restore disturbed hematopoiesis and correct complications after chemotherapy and radiation therapy of tumors, and it is also effective in the treatment of toxic stomatitis, cystitis, dermatitis, hepatotoxicity , cardiotoxicity, gastrointestinal disturbances (toxic enteritis and enterocolitis), pulmonary toxicity and neurotoxicity. When administered intrathecally, BMCP "GemaKomb" is also capable of exerting a neuroregenerative effect on organic damage to the central nervous system. In addition, the use of BMCP "GemaComb" is a biomedical way to improve the quality of HSC engraftment in bone marrow transplant and restore hematopoiesis after a course of high-dose myeloblastic chemotherapy in bone marrow transplantation.

The mechanism of action of the proposed BMCP. When the drug is administered parenterally (intravenously, intrathecally, intraventricularly, intraarterially) into the patient's body, HSCs, MSSKs and MNCs of the drug migrate to the pathology zone of damaged organs and tissues in the form of a multicellular mononuclear cluster, regardless of the etiopathogenesis of the disease: tumor, ischemia, trauma, atrophy, degeneration etc. The patotropism of the mononuclear multicellular cluster of cell systems containing HCP, MSCP, HSC and MSSK in the damaged area occurs inside the blood vessels, lymphatic vessels and / or directly inside the organ tissue and even the nervous tissue of the brain. The transfer of the cell cluster of the drug is associated with genetically determined SC homing on the gradient of inflammation concentration (SDF-a) in the pathological focus and their homing (directed migration) and cell adhesion to pathological cells. It is HSC and HKP that are the main navigation systems of the entire multicellular cluster through the vessels and inside the tissue of organs. The multicellular mononuclear cluster of the drug in the zone of maximum damage settles in the damaged organ and activates the HSC and MSSK contained in it, which then adhere to the pathological cells and, through the stander effect mechanism, affect the cellular systems of the affected organ with cytokines and cytoplasmic proteins. HSC, HKP, MSCP and MSSK, located in the multicellular mononuclear cluster of the drug, begin to exert a regulatory and sanogenetic effect on the affected cells in damaged tissues and restore their morphology and impaired POC functions. In healthy tissue, the cells of the drug are not retained and are utilized, but in the presence of damage to stationary tissues (nervous tissue, muscle tissue, articular tissue, bone tissue), the cell systems of the drug SC can stimulate regeneration by fusing their nuclei with the nuclei of POC of organs and tissues, secrete biologically active cytokines and growth factors, have a regulatory effect on POC. In tissues capable of regeneration, the cellular systems of the drug can activate the processes of reparative regeneration of organs and tissues.

Since allogeneic BMCP, if necessary, can be very effectively purified from erythrocytes and granulocytes when cultured with IL-2, its safe parenteral administration can be applied both intravenously and intraarterially, and intrathecally and intraventricularly to any patient, which makes it possible to ignore group and Rh belonging of these cells to a specific immunophenotype, group compatibility and Rh factor.

A series of additional experiments carried out by the authors of the present invention can also serve as an experimental justification for the creation of the BMCP according to the present invention. So, according to modern concepts, in the processes of metastasis, invasive growth and therapeutic resistance of malignant tumors, an extremely important role belongs to CSCs (Nessar D., Blanpain S., 2016). In a series of their own studies, the authors of the invention have shown that the mobility of human HSCs and HKPs in relation to glioblastoma cells is 61% more pronounced compared to their mobility in relation to cells of a different origin (Bryukhovetskiy I.S., Dyuyzen I.V., Shevchenko V.E. . et al., 2016). The data of the present study convincingly indicate that the migration activity of HSCs against glioblastoma cells also exceeds their migration capabilities against cells of other neoplasms, which may be associated with the high invasive exosomal potential of this tumor. However, it should be noted that the low migration activity of HSCs and HCPs in relation to lung and breast cancer cells, in comparison with that in relation to glioblastoma cells, may be due to the fact that in vivo HSCs and HCPs do not have physiological or anatomical obstacles to migration and interaction with the cells of these tumors, and chemokines produced during neoplastic growth are capable of immobilizing HSCs and HCPs from niches in the bone marrow. Of course, the difference in cultivation conditions (for example, without the addition of estrogens in the case of the MCF-7 line) affected the ability of these cells to attract HSCs, but this should not be misleading. Migration to the neoplastic focus is a fundamental property of SC of all types (Bryukhovetskiy I., Bryukhovetskiy A., Chotimchenko Y et al., 2016; Bryukhovetskiy I.S., Bryukhovetskiy A.S., Mishchenko P.V. et al., 20013; Bryukhovetskiy I.S., Duyzen I.V., Shevchenko V.E. et al., 2016). It seems to be an important moment of HSC adhesion to tumor cells of various lines. Particularly noteworthy is the phenomenon observed during monitoring in real time, when, moving along the bottom of the tablet within the field of view, the cell of glioblastoma, lung cancer and breast cancer, as it were, collects separate HSCs that attach to it (Abody KS , Braun A., Rainov, 2000). The essence of this phenomenon is that HSCs or MSSKs, introduced into a tumor or a zone of tissue ischemia, migrate after a cell that has begun to invade organ tissue, adhere to it and accompany a tumor or other pathological cell, “riding like a rider”, or "Inpiggy-backfashion" (Abody KS, Braun A., Rainov, 2000), while HSCs produce regulatory exosomes. This phenomenon was observed by the present inventors during the interaction of HSCs with glioblastoma cells, lung cancer and breast cancer. The authors consider this phenomenon to be systemic. A description of similar functions in the HSC and MSSK line in the area of ischemic stroke is available in the literature (M. Chopp et al., 2016). Moreover, it is probably advisable to speak not only and not so much about the high mobility of HSCs to OC MGB and cancer cells, but rather about their high mutual mobility with respect to each other. In this regard, it is permissible to assume that in response to hypoxic damage to the brain substance by a growing tumor or progressive ischemia, on the one hand, the processes of directed SC migration are triggered, and on the other hand, the migration of glioblastoma cells towards the ischemic areas is activated. Following this logic, it makes sense to consider the processes of invasive growth as a result of the interaction of various types of SC and OA in a hypoxic preconditioned environment. As shown in previous experiments, adhering to glial tumor cells, HSCs exchange a cytoplasmic label with them, which is manifested by the accumulation of a fluorescent dye inextricably linked with proteins of the HSC cytoplasm in lung cancer, breast cancer and MGB cells (Bryukhovetskiy I.S., Mishchenko P.V., Tolok E.V., 2014). It was shown that this process is accompanied by some suppression of the vital activity of the OK (Bryukhovetskiy I.S., Mishchenko P.V., Tolok E.V., 2014). A detailed study of the intercellular interaction indicates the high dynamics of this process. In the course of the experiment, two fundamentally opposite effects were observed in the interaction of SC and OA. In the first case, lung cancer cells predominantly accumulate a fluorescent label inextricably linked with cytoplasmic proteins introduced from HSCs, which is probably one of the mechanisms of the antitumor action of this cell type. This mechanism was used by the authors to create anticancer biological products based on HSC. In the second case, during the cultivation of HSCs with U87 glioblastoma cells, the transfer of the cytoplasmic label from OC to HSC is observed. It is important that by 96 hours after the start of the experiment in the co-culture of HSCs with glioblastoma cells, there are practically no SCs that are positive only with respect to one of the dyes used, which may indicate the recruitment of cells of this type into the neoplastic process. The question of the informational constituent of the cellular proteome, which is probably transported together with the fluorescent label from one cell type to others via exosomes, is extremely important. In this regard, the discovered effects of the transfer of a fluorescent label, inextricably linked with cytoplasmic proteins, can be a way of transferring an invasive molecular phenotype both between different types of TC and from TC into normal SC. But if MGB cells are capable of recruiting normal SC by introducing oncogenic factors into their cytoplasm, then what is the mechanism of this process? In one of the modern works, a researcher from Harvard University J. Godlewski, 2015 argues that OA secrete extracellular vesicles filled with microRNAs and providing programming of cells in contact with neoplastic cells. In addition, one of the mechanisms is the formation of nanotubes between cells, through which vesicles are exchanged (Godlewski J., Krichevsky A.M., Jonson M.D et al., 2015). There is evidence of the possibility of cell fusion or fusion of stem and neoplastic cells (Vittori M., Breznik V., Gredar T., 2016). Theoretically, this mechanism can provide the formation of a kind of "cytohybrids" with both invasive properties and valencies of SA in TC and other pathological cells. Apparently, by the example of tumors, the authors of the invention have registered the classical mechanism of the regulatory effect of HSCs on pathological cells.

Thus, the results of the experiment made it possible to draw the following important conclusions and conclusions for the creation of BMKP:

- the migration activity of HSCs and HCPs, as well as MSSKs in relation to glial tumor cells exceeds their migration capabilities in relation to TC of other types;

- HSC and MSSK have the ability to adhere to the surface of OC of various lines, and this ability is especially pronounced in relation to glioblastoma cells;

- in the process of intercellular interaction, there is a transfer of a part of the cytoplasm from HSC to OK and vice versa.

Therefore, it is theoretically absolutely true that the greater the number of HSCs and MSSKs in the BMCP, the more chances there will be to suppress the activity of the pathological cell, but the presence of a large number of HSCs also has negative consequences. Thus, a very high concentration of HSCs in the lesion focus (tumor, ischemic, degenerative, etc.) is undesirable in practice. It can be very dangerous for the patient, since with a high level of HSC cellularity in the focus and due to the rapid exchange of information microvisicles containing oncospecific TC proteins with normal SCs, normal SCs can quickly transform into CSCs and become an additional source of carcinogenesis and cause massive cancer. intoxication.

There is one more feature of GSK and MSSK, which does not allow their use without additional restrictions. When they are injected into the blood, they migrate to the patient's bone marrow after performing their regulatory functions and completing the restoration of POC. Therefore, an excessive amount of HSCs and MSSKs in the bone marrow can disrupt the balance of hematopoiesis, and it is advisable to replace some donor HSCs with MSSKs, the amount of which in the PC is not so critical. However, taking into account the strict ratio of HSCs and MSSKs in PC during mobilization of the bone marrow by colony-stimulating factors, the authors of the invention decided not to disturb this balance in the preparation administered intravenously and intraarterially into the peripheral blood.

Obviously, the purpose of using HSCs and the MSSK line in BMCPs based on mobilized PBMCs in tissue cell damage as a result of radiation or chemotherapy is to control and manage the POC functions. This is the primary and main goal of treatment with BMCPs containing HSCs, HKPs and MSSKs. Based on the obtained own experimental results of the preclinical research project at stages I-IV (report on applied scientific research on the topic "Development of a biomedical cell preparation of hematopoietic stem cells for personalized proteome-based therapy of brain tumors", carried out under the Federal Target Program "Research and Development on priority areas of development of the scientific and technological complex for 2014-2020 ", application code 2014-14-576-0057-078), as well as on the data of previous studies, it should be noted that GSK and MSSK themselves have a pronounced antitumor potential. However, this potential cannot be effectively realized only under the condition of the quantitative predominance of HSCs over other subtypes of OC. Therefore, it is very important to calculate the required dose of donor HSC and MSSK in the BMCP. The dose of the drug should be calculated on the basis of the needs for blocking CSCs, which range from 1-2% to 15% of the total cell mass of the tumor, and the number of HSCs, HKPs and MSSKs in BMCP should exceed the number of CSCs by 3 times. This calculation should be fundamental in the selection of the dose of intravenous administration. The number of HSCs and HKPs for intraventricular and intrathecal administration should also be calculated from the volume of the tumor and the amount of CSCs in it. Since the volume of the entire human cerebrospinal fluid is about 120 ml, the intrathecal administration of the drug is limited to a volume of 1-3 ml. Therefore, the dose of BMKP can be set on the basis of this calculation, but gradually, by weekly introduction of BMKP into the cerebrospinal fluid until the dose is fully set.

Taking into account the uniqueness and extremely high biological value of such a regulatory resource as HSC or MSSK, for example, in the correction of the consequences of tumor treatment (chemotherapy and radiation therapy), rehabilitation and prevention of tumor recurrence, the use of BMCP for the purpose of a regulatory effect on CSC and OC is fundamentally permissible only after therapeutic reduction of the amount of TC in the tissue of organ substances to a level of no more than 500,000 OC. That is, therapy with HemaComb should be carried out only after surgical cytoreduction of the tumor, completion of the course of chemotherapy and radiation therapy of the tumor. A practical criterion for the use of this BMCP is the impossibility of detecting a tumor focus by all standard methods of oncodiagnostics. This is due to the fact that at present, the permissible amount of OC is established for each healthy person and this amount is kept at the level of ≤500000 OC (Archakov A.I., 2012). The immune system of a healthy person is able to limit the reproduction of CSCs and OCs at this level. This figure can be adjusted, but its order is unlikely to be changed in the norm.

A no less fundamental condition for the application of the proposed BMCP is the biological certification of pathological (tumor, ischemic, etc.) cells. Let us consider the range of HSCs and MSSKs required for blocking the reproductive and proliferative functions of CSCs on various MGB subtypes. According to our own data, the number of OK CD133 + (one of the leading proneural CSC subtypes) in MHD is 1.45% of the total cell population, i.e. 7250 CSC of proneural phenotype. At the same time, according to the literature, the amount of CSCs of mesenchymal phenotype in tumor tissue removed from the brain of patients varies from 4 to 30%, i.e. 20000-150000 OK and RSK or. Taking into account that all subtypes of CSCs have the best mechanisms of resistance in anticancer treatment, then to start using BMCP, it is the number of CSCs of this type that should be ≤150000. Thus, the range of the number of CSCs of the proneural and mesenchymal phenotype in the tissue of a glial brain tumor after effective conventional antitumor therapy is from 7200 to 150,000 CSCs. It is clear that this value is conceptual in nature and in each specific case it should be set individually, depending on the molecular biological phenotype of the tumor CSC. However, the maximum value (150,000 CSCs) is the most effective criterion and the optimal amount of CSCs for intrathecal cell antitumor therapy after chemotherapy and radiation therapy has been completed.

As mentioned above and shown at the previous stages of preclinical studies, the maximum degree of inhibition of the vital functions of CD 133+ and nestin + neoplastic cells of experimental glioblastoma was observed during co-cultivation with HSC in the ratio CSC: HSC = 1: 3. In this case, it is obvious that the adequate maximum dose of HSC for creating the minimum course biodose of BMCP is the amount of HSC and MSSK equal to 4.5 × 10 ⁵ and the amount of MSSK in a volume of 1 × 10 ⁶ .

However, according to the literature, it is known that in glioblastoma up to 96% of TCs, CSCs can be (Bryukhovetsky A.S., 2013). Therefore, if we assume that these data also have a right to exist, then it is advisable to calculate the maximum dose of HSC taking this factor into account. In this case, an adequate maximum course biodose of HSC and MSSK to create a working biological dose of BMKP is the amount of HSC and MSSK equal to 1.5 × 10 ⁶ .

Thus, the specific dose of HSC must be adjusted taking into account the percentage of the CSC phenotype in a particular patient in the tumor tissue, which can be done by genetic analysis of the OC population. The range of possible use of HSC and MSSK in BMCP for prophylactic anticancer treatment ranges from 4.5 × 10 ⁵ to 9.0 × 10 ⁶ (empirically established volumes of SC in one test tube).

Таким образом, количество МНК составит 15×10⁶ клеток (15 миллионов). Если вводить ЛК в кровь, то эту дозу легко набрать путем трех доз ЛК. Но введение в ликвор и лимфатическую систему таких больших доз МНК может иметь негативные последствия. Поэтому недостающее количество ГСК в одной аликвоте ЛК с ГСК можно дополнить повторными введениями ГСК в количестве от 6,25×10⁵ до 1×10⁶ или от 50×10⁶ до 100×10⁶ МССК.The source of HKP CD34 +, CD45 +, MSSK and MSCP in BMCP is the LC of MNCs mobilized into the patient's bloodstream. A standard 1 ml aliquot of LA contains an average of 6.25 × 10 ⁵ to 1 × 10 ⁶ CD34 + MNCs. According to our own previously published data, the amount of HSC in it varies within 0.5-2%, which is largely determined by the peculiarities of the production method (from 0.5 to 3%). If we take 1.5-2% HSC as a more standard parameter of the HSC content in the cultured LK MNC, then one aliquot of HSC contains from 6.25 × 10 ³ to 1 × 10 ⁴ CD34 + HSC. To block the functions of CSCs, it is necessary to have 100 times more HSC in one dose of the drug. This dictates the need to obtain OLS in the amount

Thus, the number of MNCs will be 15 × 10 ⁶ cells (15 million). If LA is injected into the blood, then this dose can be easily collected by three doses of LA. But the introduction of such large doses of MNCs into the cerebrospinal fluid and lymphatic system can have negative consequences. Therefore, the missing amount of HSC in one aliquot of LA with HSC can be supplemented with repeated injections of HSC in an amount from 6.25 × 10 ⁵ to 1 × 10 ⁶ or from 50 × 10 ⁶ to 100 × 10 ⁶ MSSK.

Thus, the course dose of HSC and (or) MSSK was calculated for the therapy of BMCP "GemaComb", which should be implemented in 4-5 injections with an interval of one week. That is, the dose of GSK and MSSK in the preparation should be divided into 4-5 parts for a single administration. In this case, a single dose of HSC and MSSK in BMCP can be determined from the calculation of the number of injections and the maximum total dose and is 3 × 10 ⁵ HSC and MSSK per injection. Thus, to each dose of LA it is necessary to add 3 × 10 ⁵ CD34 + HSC and MSSK, which can be done by increasing the number of doses of the drug accordingly.

The use of autologous and allogeneic mobilized HSCs and HSCs in the proposed MNC in the form of MNCs of mobilized blood PCs and the MSSK line can solve the problem of insufficient effectiveness of therapies only with unmanipulated cellular systems of HSC and MSSK. The therapeutic and regulatory role of the HSC line in such drugs has been underestimated; therefore, the authors of the invention believe that the use of the proposed BMCP after radiation and chemotherapy will enhance the antitumor potential of this drug, as well as mobilize the reconstructive and restorative capabilities of the repair of damaged tissues and organs in humans.

Component A of the proposed BMCP is obtained by culturing the native bone marrow of the patient himself or a healthy donor, obtained by sternal puncture or exfusion of bone marrow punctate from the sternum in a volume of 1-5 ml. In other cases, when a significant volume of bone marrow was required, allogeneic or autologous bone marrow biomaterial was obtained during the operation of bone marrow exfusion performed under spinal or epidural anesthesia by trepanbiopsy of bone marrow from the sternum and iliac bones of a patient or biocompatible donor and took up to 400-1000 ml of bone marrow. Subsequently, the total mass of cells was fractionated from the obtained bone marrow or LA PC by gradient centrifugation, and an MNC suspension was obtained. In some cases, the source of the MSSK line was the adipose tissue of a patient or a healthy donor obtained by its surgical excision or obtained by a standard liposuction procedure. Subsequently, the KM or LK MNK PK exfusate was cultivated in a GMP-standard clean room in culture boxes. Adipose cells were cultured under similar culture conditions to obtain MSSK. The MSSK line was isolated after 3-5 passages of bone marrow cells.

The MSSK line is made from 10-50 ml of bone marrow exfusate or leucoconcentrate MNC PK.

Isolation and cultivation of the MSSK line

MSSCs were isolated and isolated from various sources according to well-known international protocols - from bone marrow (P. Penfornis, R. Pochampally et al., 2011) from umbilical cord blood (Can A. and Balci D., 2011) or from adipose tissue (Zachar V ., Rasmussen JG and Fink T., 2011) of the patient or donor. To isolate MSSK from adipose tissue, a domestic modification was used (Teplyashin A.A., 2012), and to isolate and isolate MSSK from the bone marrow of umbilical cord blood, a modification of Professor A.G. Konoplyannikov was used. (2014).

Bone marrow harvesting material was used to obtain MSSK. Exfusion of 5-50 ml of bone marrow from the iliac crest was carried out with special biopsy needles for trepanbiopsy.

An example of ISSC isolation from bone marrow

After settling the bone marrow sample for 1-2 hours at room temperature, the isolated upper cell fraction was taken with a Pasteur pipette and resuspended in the growth medium. As a growth medium, we used RPMI-1640 medium containing penicillin-streptomycin (100 U / ml), amphotericin (100 ng / ml), L-glutamine 2 mM, 20% fetal calf serum. The cultivation was carried out in plastic flasks with a bottom area of 25 cm ² , into which (2-10) × 10 ⁵ cells / cm ² were introduced into 8 ml of growth medium. The vials were placed in a CO ₂ incubator (5% CO ₂ ). After 48 hours, the medium with non-adherent cells was removed and replaced with a fresh one. Upon reaching a confluent monolayer (90-100%), the cells were subcultured using 0.25% trypsin-EDTA solution into new flasks, first with a bottom area of 75 cm ² , and subsequently, with an increase in the cell mass, into large culture flasks with a bottom area of 175 cm ² ... This method allows by the end of 5-6 weeks to achieve a population of MSSK in the amount of (1-2) × 10 ⁸ cells, sufficient for transplantation into the recipient organism. The study of specific markers of the obtained MSSK cultures by flow cytofluorometry shows that they belong to the population of CD10 ^low , CD34-, CD45-, CD44 +, CD90 +, CD105 +, CD13 +, CD117-, which is characteristic of cells derived from multipotent mesenchymal SCs of an adult organism.

The entire cell mass of MSSK in the amount of (1-2) × 10 ⁸ cells undergoes a transcript-modifying pretreatment by conditioning the MSSK culture with one of the four perturbogens at a certain concentration in micromol / L (μM): Sol. Propidium iodide (0.000006 μM), Sol. Acidi acetylsalicylic (0.0001 μM), Sol. Trichostatin A (0.0000001 μM), Sol. Niclosamide (0.0000122 μM). After 6 hours, the medium is removed and replaced with 0.9% saline NaCl. The obtained cellular biomaterial is washed from the culture medium with the same 0.9% saline NaCl solution by double centrifugation of the cells at 2000 g, and the material can be immediately used immediately for transplantation. It is washed from the culture medium with saline and transferred into 200 ml of saline with 10 units / ml of heparin. If the material is planned for intrathecal administration, then it should be packaged in aliquots of 1 × 10 ⁶ MSSK into cryovials of 2 ml and frozen in a program freezer. If the material will be used for intravenous or intra-arterial administration, then the entire volume (1-2) × 10 ⁸ MSSK will be cryopreserved in cryovials of 5-10 ml or special bags for storing bone marrow.

Cryopreservation of cell material

1. Sterile dimethyl sulfoxide (DMSO) solution is added to a cooled suspension of cells in autologous plasma to a final concentration of 10%.

2. The suspension is packaged in the required number of cryovials, marked, packaged and frozen.

3. After freezing, the cryovials are transferred to a Dewar for storage under nitrogen vapor.

Using cellular material for transplantation

1. The material intended for transplantation is washed from the culture medium with physiological saline and transferred into 200 ml of saline with 10 units / ml of heparin.

2. Transportation of cell material is carried out on ice in a thermally insulated container.

3. The cellular material is injected intravenously using a transfusion system for 1 hour. 10 minutes before the start of the procedure, the patient receives 4 mg of metipred.

An example of MSSC isolation from adipose tissue

Adipose tissue was taken by biopsy in a volume of 10 g. The obtained 10 g was washed three times with PBS (Gibco) at pH 7.2, without Ca2 + and Mg2 + ions, containing a single solution of antibiotics and antimycotics (Gibco), the final concentration of penicillin is 100 U / ml, streptomycin 100 μg / ml, amphotericin B 0.25 μg / ml, and impurities of dense connective tissue are removed. Then mechanical fragmentation of the tissue is carried out with medical scissors in culture dishes with a diameter of 10 cm (Costar) until a finely dispersed mass is obtained, which is transferred into two 50 ml tubes with a conical bottom (Costar) and each sample is suspended in 25 ml of DMEM medium containing antibiotics and antimycotic. Next, a stage of enzyme treatment is carried out: 1 ml of a 2% type I collagenase solution (Gibco) in a PBS buffer solution without Ca2 + and Mg2 + ions is introduced into the resulting suspensions, to a final enzyme concentration of 0.075%, incubated at 37 ° C for 30 minutes at slow rocking. The resulting mixture is thoroughly mixed, then an equivalent volume of DMEM containing 10% FBS, antibiotics and antimycotic is added and centrifuged for 10 minutes at 1000 g. The supernatant and fat droplets are removed, the precipitates are combined and suspended in 10 ml of cold lysis buffer (+ 4 ° C) containing 155 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM Na ₂ EDTA. The mixture is thoroughly mixed and incubated for 3-5 minutes at room temperature. Next, 10 ml of DMEM medium containing antibiotics and antimycotic is added to the cell suspension. The cells are pelleted by centrifugation for 10 minutes at 1000 g. The supernatant is removed and a washing step is carried out. The cell pellet is resuspended in 30 ml of DMEM medium containing antibiotics and antimycotic, and centrifuged at 1000 g for 10 minutes. The resulting precipitate is resuspended in 25 ml of DMEM medium containing antibiotics and antimycotics. The resulting cell suspension is passed through a filter with a pore size of 100 μm and centrifuged for 10 minutes at 300 g. The pellet is resuspended in DMEM supplemented with antibiotics and antimycotics, 10% FBS and 25 ml of a single solution of non-essential amino acids (Non-Essential Amino Acids, Gibco). The suspension is passed through a 10 µm filter. The result is a homogeneous cell fraction, free of cellular debris and blood cells.

The resulting cell suspension is evaluated by counting in a Goryaev chamber and seeded into vials with an area of 75 cm ² at the rate of 106 cells / cm ² . The percentage of adhered cells is approximately 1-1.5%. Thus, the amount of mesenchymal SC isolated from 1 g of adipose tissue is about (1.5-3) × 10 ⁴ cells. After 24 hours, the medium is changed to DMEM containing antibiotics (100 U / ml penicillin, 100 μg / ml streptomycin, Gibco), 10% FBS, and a single solution of nonessential amino acids (Non-Essential Amino Acids, Gibco). Upon reaching a monolayer, the cells are subcultured, a visual assessment of the cell morphology by phase contrast microscopy is performed, the mitotic index and the time of cytogenesis are estimated. According to the results of morphological analysis, two subpopulations were identified in the cell fraction obtained after isolation. The first type of cells is a subpopulation of small spindle-shaped cells 10-15 microns in diameter with a clearly defined nucleus and homogeneous cytoplasm. The second type is represented by cells of a rounded shape with a flat outgrowth of the cytoplasm elongated on one side, the cell size reaches 40 microns; there is a dark nucleus displaced to one edge, heterogeneous cytoplasm and increased granularity in the nuclear region. In the phase of logarithmic cell growth, the mitotic index and the time of cytogenesis are calculated. The ratio of the number of mitotic cells to the total number of cells is 34%. Doubling time 54-62 hours. The resulting cells are immunophenotyped by indirect immunofluorescence. A flow cytometer (Beckman Coulter) revealed a high level of expression of the following antigens CD10, (CALLA), CD 13 (APN), CD44 (hyaluronic acid receptor), CD90 (Thy-1), CD 105 (endoglin) (Becton Dickinson). The absence of expression of HSC markers CD34, CD45, and CD117 (Becton Dickinson) was also shown. The results of immunophenotyping show that the population of the obtained cells corresponds to MSSK in terms of the expression of surface antigens.

Component B was isolated from fresh own or donor bone marrow exfusate or LK MNC PC using leukocytopheresis technology (Miltenyi Biotec, Germany or Spectra Coba, USA). Subsequently, for 3-7 days, the amount of PB MNCs was increased by cultivation in a medium that blocks their differentiation, but promotes an increase in the amount, and then washed from the culture liquid by double centrifugation with 0.9% CRNC, divided into aliquots of 1 × 10 ⁶ HSCs were both cryopreserved and stored at -196 ° C.

Obtaining LA MNC from unmanipulated bone marrow

Requirements for bone marrow donors

Donors should be screened for HIV 1 and HIV 2 infections, immunodeficiency syndrome; syphilis, hepatitis B and C, acute infectious diseases, prions.

Getting bone marrow

The sampling of material for the subsequent isolation of cells is carried out by a surgeon who has practical experience in carrying out this procedure. Bone marrow exfusion is performed in the operating room by puncturing the distal iliac crest under general or local anesthesia. The puncture is performed with a sternal needle, the bone marrow is taken with a Luer-type syringe, the bone marrow suspension is transferred into a bag with isotonic anticoagulant.

The main stages of bone marrow exfusion

1. Perform a thorough disinfection of the skin in the distal iliac crest.

2. A sternal needle, equipped with a mandrel and a protector, is injected vertically until it stops in the bone.

3. The bone wall is pierced with a uniform rotary motion. Penetration of the needle into the bone marrow space is determined by the weakening of the resistance provided by the cortical layer of the bone.

4. After puncturing the bone wall, the mandrel is removed, a precisely fitted syringe (20 ml) containing 100 IU heparin / ml of bone marrow is placed on the needle and the piston is pulled.

5. After filling the syringe with the required amount of bone marrow, the resulting exfusate is transferred into the bag of the donor system with isotonic anticoagulant.

6. After changing the position of the needle, if necessary, repeat the exfusion until the required amount of bone marrow is obtained.

7. If necessary, take peripheral (venous) blood using 20-50 ml syringes or a bag of the donor system.

8. The sample is accompanied by documentation containing information characterizing this sample (donor data, identification code, source, time and conditions of collection, storage conditions).

9. The obtained sample is delivered in a sterile mixing box and an isothermal transport container to the laboratory for the isolation of MSSK.

Obtaining LK MNC mobilized PC

Mobilization of GSK in PC

In order to increase the number of SCs in the PC, the donor or patient receives 8 injections of G-CSF subcutaneously with an interval of 10-12 hours for 4 days. G-CSF is a medicinal product obtained by genetic engineering and is an absolute analogue of the human factor. In the first three days, the dose of the drug is 2.5 μg / kg, on the last day, the dose is doubled. A general blood test is performed daily and an abdominal ultrasound scan and fibrogastroscopy are performed on day 4-5.

Collection of GSK and GKP

Collection is carried out 5 days after the start of G-CSF stimulation on a COBE-Spectra blood separator using a disposable separation system and standard solutions. The duration of the procedure is 3-4 hours depending on the speed of the procedure, donor weight and blood test parameters. The procedure is carried out by taking blood from one vein, processing it inside the separator, taking a certain amount of SC and returning the rest of the blood components to the donor through another vein. Venous access is carried out by puncture of two peripheral veins or through a double-lumen central catheter inserted into the subclavian vein during the session. The average volume of collected material is 300-400 ml. The collected material is assessed by two parameters - the total number of nuclear cells in the separator and the number of CD34 ⁺ cells per kilogram of patient weight. Nuclear cells in the separator are determined by counting in the Goryaev chamber prior to any manipulation. The percentage of CD34 ⁺ cells in the cell suspension obtained during cytopheresis is determined by flow cytometry.

Definition of peripheral SK and HKP

The determination of the subpopulation composition of CD34 + cells is carried out cytofluorimetrically using the triple labeling method (simultaneous staining of cells with antibodies to three different antigens loaded with different fluorochrome dyes).

Drug standardization

Determination of the number of progenitor cells is carried out cytofluorimetrically, in a direct reaction of immunofluorescence (RIF). A double-labeling method is carried out, with simultaneous staining of the cell substrate with monoclonal antibodies to the CD34 + antigen as the main marker of the cells of the HSC pool and to the CD45- molecule, the leukocyte antigen that determines HSC. This technique allows you to immediately calculate the ratio of the number of CD34 + cells and all HSCs (CD45 +) in the material. To assess the levels of non-specific binding, a portion of the cells are stained with isotypic controls. Mouse IgG1 isotype immunoglobulins (IgG1) labeled with dyes similar to the used monoclonal antibodies (PE, FITC, PerCP) are commonly used as isotypic controls.

Cell sample preparation

Before staging the reaction, the cells of the PK and the cytopheresis product are freed from the impurity of erythrocytes by a standard lysis procedure and subsequent washing in PBS-BSA by centrifugation at 1000 g for 5 minutes. PBS-BSA can be replaced with Hanks or 199.

Erythrocyte lysis technique

1. Add 2 ml of lysing solution to 0.2-0.5 ml of cell sediment, mix, incubate until the solution becomes transparent (varnish).

2. Wash the cells twice with medium 199 during centrifugation (1000 g, 5-7 min).

With the isolated cells, direct RIF is carried out in a 96-well plate, while a panel of three wells is used to determine the number of cells in any material - 1) unstained cells; 2) cells stained with isotypic controls with a label corresponding to the label of the used monoclonal antibodies (MCA); 3) cells stained simultaneously with MCA to the CD34 and CD45 antigens.

It should be noted that anti-CD34 + mAbs are preferable to use with phycoerythrin (PE) or peridinine chlorophyll (PerCP) tags. These fluorochromes have a higher specific signal level compared to fluorescein isothiocyanate (FITC).

To determine the number of CD34 + cells, antibodies to the CD34 antigen of the HPCA-2 clone (8G12), the IgG1 isotype, are optimal.

Thus, the standard panel looks like

1.control IgG1 PE + control IgG1 FITC

2.control IgG1 PE + MCA to CD45 FITC

3. MCA to CD34 PE (NRSA-2) + MCA to CD45 FITC

RIF production

1. In the wells make cells in an amount of at least 500,000 per well.

2. Next, a cocktail of antibodies is added to each well in accordance with the panel and gently resuspended with a pipette. Each MCA is taken in an amount of 10 μl per well, the total volume of MCA in the well is 20 μl.

3. Cells are incubated with antibodies for 30 minutes at 4 ° C (bottom shelf of a conventional refrigerator).

4. At the end of the incubation time, the cells are washed twice to remove unbound antibodies by centrifugation for 5-7 minutes at 1000 g.

5. The cells are transferred into special plastic tubes for counting on a flow cytometer.

6. The volume of the cell suspension in each tube is adjusted to 200-500 μl by adding PBS-BSA to the cells.

The score must be made immediately after the reaction has been set. Counting and recording on a flow cytometer

The reaction is assessed using a 5-parameter flow cytometer. This scheme is applicable to any configuration of a flow cytometer. CD34 + cells in PK represent a small cell population. Even under conditions of preliminary stimulation of hematopoiesis, the maximum percentage of these cells is 1-3%. Therefore, when counting these cells, at least 20,000 cell events should be accumulated in each analyzed sample.

Cell collection and analysis is performed in the CD45 + cell gate, which includes all HSCs. In this context, the gate means the area of accumulation of events, limited by certain parameters. In this case, SSC / FL-1 (CD45 FITC). That is, all CD45 + events will be visible on the abscissa axis (FL-1) on the dotted cytogram. Along the ordinate (SSC parameter — lateral light scattering), cellular events will be ranked according to their granularity (cytometric, not morphological term). The absolute number of CD34 + cells in 1 μl of blood and cytoconcentrate was calculated based on the number of leukocytes in the hemogram on the day of the study.

Principle of determination of CD34 + cells in hematopoietic tissue. Sample recording

1. Choice of the area of analysis - gate. The instrument has a menu for recording cell samples Acquisition. At the first stage, in the Setup mode (mode of viewing a cell sample without recording), samples # 1 (IgG1 PE + IgG1 FITC) and # 2 (IgG1 PE + CD45 FITC) are scanned and CD45 + events are detected. These events are located to the right of 10 ¹ values (the average threshold value of the specific signal level for fluorescein FITC) on the abscissa - FL-1 (CD45 + cells) and are quite clearly visualized in comparison with the control sample No. 1. By sample 1, the entire area located to the right of the main cell density is limited - the gate, which includes only CD45 + events in sample 2 and contains the minimum number of cellular events in sample 1.

2. The device is switched to the sample recording mode (normal), and sample # 1 is collected and recorded for 20,000 cell events without a gate (the entire cell population). The recording of this sample in the gate does not make sense, since it does not contain MCA to the CD45 antigen. As shown above, this trial is necessary to correctly select a gate containing only CD45 + events.

3. Samples # 2 and # 3 are collected and recorded in the CD45 + gate. This gate is identical for these samples (that is, the gate parameters are not changed during collection). The minimum number of cellular events is 20,000 for each sample.

Analysis of recorded samples

1. Go to the Analysis menu of the recorded samples. Open the cytogram (dot-plot) of sample 2 in the SSC / FL-2 parameters, in the R1 gate - CD45 + (the gate was selected by us earlier at the time of the collection recording and was automatically transferred to the analysis mode). In this case, the ordinate will reflect the SSC parameter - the granularity of the events that fell into the analyzed gate, and the abscissa FL-2 - the detector reflecting the levels of nonspecific binding for the phycoerythrin dye detected by antibodies to mouse IgG1PE immunoglobulins.

2. A control marker is set according to this cytogram so that there are practically no cellular events in the lower right quadrant. On average, for the vertical marker bar, the values will be about 130 (to the right of 10 ² ), and for the horizontal marker bar, the average values will be 60, which corresponds to the concept of SSC-low. That is, cells with a minimum number of cellular inclusions. Thus, the levels of non-specific binding will be equal to or close to zero.

3. Automatically go to sample # 3. All instrument readings taken for the second sample are retained. That is, the cytogram is opened in the SSC / FL-2 parameters, the gate selected earlier when collecting for CD45 + cell events is saved, and the values of the control marker established by sample 2 are saved. Only on this cytogram on the abscissa axis will be reflected not the levels of nonspecific binding, as in the second sample, but specific CD34 + events. The percentage of CD34 + cells is displayed by the device automatically.

4. If necessary, depletion of the obtained LA from αβ T cells (lymphocytes) was performed on a standard CliniMACS® plus apparatus for immunomagnetic cell selection (equipment for clinical use) using special CliniMACS TCR alpha / beta Kit antibodies according to the manufacturer's standard protocols. The cultivation of depleted LA was carried out as described above.

Cryopreservation of LK MNC

The traditional cryopreservation method is to add DMSO to the cell suspension at a final concentration of 10% and standard freezing at 1 degree per minute to -80 ° C or -120 ° C using standard electronic software freezer programs, followed by storage in liquid nitrogen or liquid nitrogen vapor ...

Isolation of the cell fraction

Hematopoietic progenitor cells (HCP). The separated cells are concentrated by centrifugation at a centrifuge speed of 2000 rpm per minute for 10 min at + 18 ° C. With the help of a manual plasma extractor, plasma is removed from the container as much as possible, the cells remain in a volume of 40-60 ml.

Adding a cryoprotectant

Highly purified DMSO is used as a cryoprotectant for hematopoietic cells. To the resulting cells with constant stirring add equal volumes of polyglucin and DMSO. Mixing DMSO with polyglucin occurs as an exothermic reaction with the release of a moderate amount of heat. The concentration of DMSO in polyglucin is 10-12%. Thus, its final concentration in the material to be frozen will be 5-6%.

The use of polyglucin made it possible to reduce the amount of DMSO by half, to 5-6% of the final concentration, since polyglucin has the following properties - firstly, the ability to disaggregate cells and thereby improve the penetration of cryophylactic into cells, and secondly, polyglucin (6% dextran) itself is a cryophilicist.

Counting the number of cryopreserved cells

At the next stage, it is necessary to count the nucleated cells, as well as CD 34+ cells to be frozen.

Selection of the optimal amount of material to be frozen

In cryopreservation, in order to create the best conditions, the ratio of the volume of the plastic container to the volume of the material frozen in it and the concentration of the frozen cells are important. It was found that the optimal concentration of frozen cells is (40-100) × 10 ⁶ cells per ml.

Freezing the LC of mobilized MNCs

Depending on the final volume of the material to be frozen, the number of polymer ampoules (20-25) is selected for deep programmed freezing. Next, the cell suspension is transferred into these heat-resistant plastic tubes.

At present, the methods of cryopreservation of cell preparations involve the use of electronic program freezers. To freeze ampoules with cell suspension, they are placed in an electronic program freezer. The cooling rate of biomaterial at temperatures from 0 ° C to -40 ° C is 1.1 degrees per minute, and then 1 degree per minute to -80 ° C or -120 ° C. After the completion of the programmed electronic freezing cycle, the tubes with the frozen material are transferred to the storage for long-term storage. Then the tubes with the material to be frozen are placed in liquid nitrogen vapor at a temperature of -165 to -170 ° C, where they will be in liquid nitrogen or its vapor until use.

The proposed method takes into account and implements the main requirements for the biomaterial subjected to cryopreservation, namely, the maximum preservation of the viability of the SC, the minimum volume (100-120 ml) of the material to be frozen with the maximum number of cells contained (up to 100 × 10 ⁶ / ml), for PC minimum volume 50-60 ml; the minimum amount of autograft volume.

The advantages of cryopreservation in liquid nitrogen vapors are greater safety of storage of test tubes with cellular biomaterial and significantly lower nitrogen consumption;

Cultivation

The liquid culture system consists of Pskov's medium modified by Dulbecco (Iscove's modified Dulbecco's Medium IMDM, Gibco, UK) with the addition of 0.5 g / L bovine serum albumin (Sigma Aldrich), 0.39 μg / ml human insulin and 60 μg / ml transferrin (Gibco). Stem cell growth factor SCF, also called c-Kit-ligand (c-KitL, R&D Systems), at a concentration of 100 ng / ml, and Flt3-ligand (Flt3L) at a concentration of 20 ng / ml (R&D Systems ) and ionomycin (Sigma Aldrich) at a concentration of 0.1 μM / ml

Method of cultivation of LA MNC PC. The cells are cultured at 37 ° C in a humid atmosphere with an influx of 5% CO ₂ . Fresh medium and cytokines are added every third day. The cultivation time is 3-7 days. The used growth factors contribute to the proliferation of SC, and there is no need to add agents that suppress differentiation. The resulting SC suspensions of any individual can be stored frozen or can be introduced into BMCP immediately after cultivation and applied to patients.

Conclusion: The material is intact. Based on flow cytometry data, lymphocytes possible for analysis are 84.7%. Among them, mature T-cells account for 81.2% with a predominance of CD4 + subpopulations, γδ-T-cells accounted for 1.2%. Heterogeneous NK cells are represented by populations of CD94 +, CD16 +, CD56- and CD94-, CD56 +, CD16- and CD16 ++, CD56 ++, which in total make up 14.0% of lymphocytes. Populations CD16 +, CD56low not identified

Obtaining the proposed BMCP component, which allows the cultivation of SCs with cytokines that promote proliferation, but not differentiation of SCs, opens up prospects for the use of a patient's own SCs for the treatment of diseases such as coronary heart disease, burns, long-term non-healing wounds and injuries of the extremities, spinal and head injuries brain. Autologous SCs can be isolated and stored frozen for emergency use. At the same time, the ability for subsequent differentiation in such cultured cells is preserved at the level of initially secreted SC.

Cryopreservation of cell material

A sterile DMSO solution is added to a cooled suspension of cells in autologous plasma to a final concentration of 10%. The suspension is filled into the required number of cryovials, labeled, packed and frozen. After freezing, the cryovials are transferred into a Dewar vessel for storage in nitrogen vapor.

Using cellular material for transplantation

The material intended for transplantation is washed from the culture medium with physiological saline and transferred into 200 ml of saline with 10 units / ml of heparin. Transportation of cell material is carried out on ice in a thermally insulated container. The cell material is injected to the patient intravenously using a transfusion system for 1 hour. 10 minutes before the start of the procedure, the patient receives 4 mg of metipred.

Component C. The proposed BMCP was made ex tempore by mixing under sterile conditions the cells of component A, thawed in a water bath, with component B and component B in the form of 0.9% saline NaCl solution or (and) a heterogeneous biopolymer matrix, for example, the SpheroGel matrix.

The proposed BMCP "GemaComb" in saline solution can be injected subcutaneously, intramuscularly, intravenously, intraarterially, intrathecally, and on the basis of the indicated matrix - into the tissue parenchyma by interstitial injections and implantations.

The invention allows the use of BMCP "GemaComb" as a therapeutic and prophylactic antitumor agent of choice in the rehabilitation of cancer patients after radiation therapy and chemotherapy, in patients with damage and diseases of the central nervous system, in systemic diseases and degenerative processes in organs and tissues, in organ and polyglandular insufficiency , as well as to improve the quality of life of gerontological and somatically weakened patients and prolong their life.

Example. Data of phenotypic analysis of MNCs in one representative experiment carried out on LK cells of donor N.

Preparation of BMCP for transfusion and its application. Defrosting components

Defrosting of components A and B is performed immediately before transplantation or transfusion in a treatment room in a water bath at a temperature of 37-40 ° C until the frozen material passes into the liquid phase. Subsequently, by centrifugation at 1500 rpm, each component used is deposited on the bottom of a centrifuge tube, the supernatant is sucked off and 1 ml of 0.9% saline NaCl is poured or transfused into a heterogeneous biopolymer collagen matrix. The procedure is repeated twice. Defrosted components A and B of the preparation can be used within 6 hours after preparation. If the drug has not been used during this time, then it must be disposed of.

Intravenous or intra-arterial transfusion of BMCP "GemaKomb"

Intravenous or intra-arterial administration of BMKP is performed by standard intravenous or intra-arterial slow bolus administration of the drug from one to 20 biodoses, diluted in 0.9% saline NaCl.

Intrathecal introduction of BMCP

Intrathecal administration of the drug "GemaComb" should be carried out only in the intensive care unit. The drug is transfused through a lumbar puncture made in a typical place (in the L3-L4 gap) under local anesthesia with 1% lidocaine solution. Then, 3 ml of cerebrospinal fluid is taken and mixed with one or two biodoses of the cell preparation (maximum volume up to 3 ml), the cell preparation is resuspended in the cerebrospinal fluid and slowly injected into the subarachnoid space. Apply an aseptic bandage. If necessary, repeat intrathecal transfusions of the drug "GemaComb", but not earlier than 7 days after cytotransfusion. To correct possible allergic reactions with a prophylactic purpose, a single intramuscular injection of corticosteroid drugs (4 mg of dexamethasone) is done.

The main criteria for the effectiveness of the intervention is the improvement of clinical symptoms. The term of the expected result is extremely individual for each patient and depends on the volume of organ damage, the duration of the injury, the degree of compensation for the impaired functions. The effectiveness of therapy varies from 1-3 days to 12-16 months after transfusion, and is assessed by clinical scales and electrophysiological research methods (ECG, cerebral EEG mapping, transcranial magnetic stimulation, somatosensory evoked potentials and electroneuromyography, as well as complex urodynamic research, etc. .).

Possible complications and ways to eliminate them

There was not a single fact of complications as a result of infusions of BMCP "GemaComb" into the blood and cerebrospinal fluid of cancer patients, which is associated with the use of autologous biomaterial and immunocompatible allogeneic material, selected according to the immunophenotype of blood. However, theoretically, allergic reactions to cryoprophylaxis of DMSO are possible in case of insufficient washing before use. Treatments for these allergic reactions are standard. To prevent possible allergic reactions of the patient to the drug transfusion, it is advisable to make a single intramuscular injection of the drug "Dexometasone" at a dose of 4 mg / ml 20 minutes before the manipulation.

The effectiveness of BMKP "GemaComb" in the treatment of cancer

The analysis of the efficacy of the "GemaComb" preparation was tested on various groups of cancer patients and compared with the efficacy of conventional LA of mobilized HSCs used to restore hematopoiesis during bone marrow transplantation.

Characteristics of adult cancer patients (group 1). The average age of adult patients was 33 years (median 32 years, from 16 to 64 years), the weight of patients varied from 43 to 113 kg. Among the patients, men prevailed - 113 people, women - 54 people (32.3%). The distribution of patients is shown in Table 2.

The experimental group was dominated by patients with oncohematological pathology (158 people, 94.6% of the total number of patients). A small number were patients with non-hematopoietic tumors (only 5.3%, 9 people). In all cases, G-CSF was used to mobilize CCM in adults. Pre-treatment in adult patients ranged from 2 to 22 courses of chemotherapy. The chemotherapy regimens preceding the administration of growth factors varied depending on the patient's diagnosis. Thus, in the case of Hodgkin's lymphomas and in several cases of NHL, the DEXA-BEAM and BEASOPP regimens were used. In some cases of B-cell lymphomas in adults, rituximab regimens (R-CHOP) have been used. CHOP regimens have also been used in several cases for Hodgkin's lymphoma. In multiple myeloma, the prescription of growth factors was preceded by the VAD regimen, in breast cancer - by CAF. 22 patients underwent radiation therapy.

Characteristics of donors for the preparation of an allogeneic form of the drug

For the preparation of the "HemaComb" preparation, 61 samples of LA were used, obtained from 50 donors during the collection of hematopoietic tissue for allogeneic transplantation to 47 patients. Before collection, donors were mobilized with HSC using a standard stimulation protocol in mono-mode: growth factors without chemotherapy (G-CSF on the first - third days at 5.0 μg / kg body weight twice, on the fourth day at 10.0 μg / kg body weight twice). The most frequent transplantation was from mother to daughter (19 procedures), from mother to son - 14 transplants, from father to son - 9 transplants and from father to daughter - 5 transplants. The choice of a donor for related transplantation was primarily determined by compatibility, health status of potential donors, and age. Other things being equal, for the father and mother, preference was given to the mother in view of the existence of the phenomenon of "fetomaterin chimerism". The average age of donors was 34 years (from 17 to 48 years), women predominated (36 women and 14 men).

51 SC transplants were performed, of which 31 were haplografts (partially compatible) and 20 allogeneic related transplants. Restoration of hematopoiesis and manifestations of toxicity after chemotherapy and radiation therapy was carried out on the basis of BMCP "GemaComb" in 164 patients.

A set of material for a full-fledged transplantation (that is, at least 2.0 × 10 ⁶ CD34 ⁺ cells per kg of body weight of the recipient) for most patients was carried out in 1 session of leukopheresis.

For 8 patients, the material for complete transplantation was collected in 2 sessions of leukopheresis; in one case, 3 consecutive procedures for collecting SC were required. For 7 patients, the number of transplanted CD34 ⁺ cells was less than 2.0 × 10 ⁶ per kg of the recipient's body weight (from 0.1 to 1.93) × 10 ⁶ . However, the transplants were performed.

Immunological characteristics of the drug for bone marrow transplantation and BMCP "GemaKomb"

The assessment of the number of SCs and the characteristics of their subpopulations were carried out in the PC samples on the eve of the cytopheresis procedure and directly in the samples of the leukopheresis product. In most patients, the amount of HSCs in PC samples was assessed on the eve or on the day of the first leukopheresis procedure.

Assessment of the quality of the transplant material was carried out immunologically in the reaction of direct immunofluorescence, taking into account the data on a flow cytometer.

Immediately after the leukopheresis procedure, the samples were counted for CD34 ⁺ cells, as well as for mature T cells (CD3 ⁺ ); in some donors, the number of T lymphocyte subpopulations (CD8, CD4) and the number of NK cells (CD56) were also assessed.

The estimation of the number of CD34 ⁺ CK was carried out on the basis of the ISHAGE protocol in the reaction of double immunofluorescence using direct conjugates of monoclonal antibodies to the CK antigen CD34 and the general leukocyte antigen CD45. Fluorochrome labels and clones of the antibodies used are shown in Table 3.

To assess subpopulations of CD34 ⁺ cells, including those non-expressing the total leukocyte antigen, the ISHAGE protocol was modified. The number of MCSC CD34 ⁺ was calculated for the entire cell population of the sample, and then the obtained percentage of CD34 ⁺ cells was recalculated for leukocytes (CD45 ⁺ cells) depending on their number in the sample. The subpopulation composition of HSCs was assessed within CD34 ⁺ cells using a triple fluorescent label.

The proportion of early precursors for the expression of linearly unrestricted antigens (HLA-DR, CD38, CD71), markers of early stages of differentiation (CD117, CD90), and cell adhesion molecules (CD50, CD56, CD45) was estimated. As well as the number of linearly committed HSCs: myeloid (antigens CD13, CD33), lymphoid (CD7, CD19) megakaryocytic (CD61) and erythroid precursors by expression of the antigen of the cells of the red series of glycophorin (CD236a).

Based on the antigens analyzed in the work, the direction of differentiation was characterized with a high degree of probability and the number of pluripotent and bipotent (myeloid CD13 ⁺ , CD33 ⁺ ) or lymphoid-committed (B-line - CD19 +, CD34 +, T-line - CD7 +, CD34 +) hematopoietic stem cells, as well as SCs with megakaryocytic differentiation (CD61 +).

The population of progenitor cells of erythroid direction of differentiation (CD71 +, CD236a +) among the samples of this analyzed group was very small and the analysis of this subpopulation was not carried out.

Statistical processing of the obtained data was carried out using SPSS version 17 for Windows. When processing the material, the frequency function was used to describe sequential series of variables, mean values, median, standard error of mean values and variability of the values of variables, the function of bivariant correlations to determine the correlation coefficient of two independent variables (the correlation is reliable at p≤0.05), the function of comparing means values of independent and dependent variables (confidence interval 95% and higher), the function of encoding variables in accordance with the intervals of values of the selected variable.

The results obtained showed that the best stimulation effect based on the CD34 count⁺ leukocytes were observed in patients with multiple myeloma and NHL in comparison with Hodgkin's lymphoma. Samples with maximum CD34 count⁺ leukocytes were characterized by a lower proportion of CD34⁺CD38⁺, CD34⁺CD33⁺and CD34⁺CD7⁺ CCM. In 35 cases, only PC cells were used as transplant material in adult patients (30 patients - 30 first transplants and 5 repeated ones; the dose collected in one leukopheresis cycle was enough for repeated transplantation in patients with multiple myeloma, in accordance with the clinical protocol). In 9 cases, as a hematological support, transplantation of PC cells was carried out in the form of the drug "GemaComb". In 30 cases, only BMKP "GemaKomb" was used.

In general, in the group of patients who received only peripheral SCs, the average rate of platelet recovery up to 20,000 cells / μL was 14.26 ± 1.14 days, with a median of 12 days and a range from 9 to 59 days. The average rate of neutrophil recovery up to 500 cells / μL was 12.2 ± 0.3 with a median of 12 days and a range from 7 to 24 days.

The terms of hematopoiesis recovery in 30 patients who received GemaComb were shorter than with conventional transplantation (leukocytes - 12 days (BMCP GemaComb) versus 14 days (umbilical cord HSCs), platelets 14 days (BMCP “GemaComb”) versus 18 days ( The differences are statistically insignificant, most likely due to the difference in the number of cases - 35 pCK transplants versus 30 BMCP “GemaComb”.

Reliably faster recovery of neutrophils was revealed when using BMCP "GemaComb" in comparison with transplantation of a part of recruited HSCs, despite a lower transplanted dose of HSCs (3.5 × 10 ⁶ / kg versus 6.2 × 10 ⁶ / kg, p = 0.001).

Shorter periods of critical thrombocytopenia are associated with a higher dose of CD34 ⁺ CD71 ⁺⁺ and CD34 ⁺ CD13 ⁺ HSCs (p <0.03), a shorter period of critical neutropenia was noted with a greater proportion of CD34 ⁺ CD71 ⁺⁺ HSCs and CD34 ⁺ CD19 ⁺ HSCs ( p <0.035), which was typical for the use of the drug "GemaComb". Shorter periods of thrombocytopenia were due to the low content of CD34 ⁺ CD61 ⁺ CCM in the first day of collection. The efficacy of BMCP "HemaComb" for the correction of critical thrombocytopenia was obvious and statistically significant (p <0.05) compared with conventional cell transplantation.

The optimal total dose of transplanted HSCs in BMCP "GemaComb", providing in adult patients the recovery of neutrophils and platelets within no more than 11 days from the moment of transplantation, is (3.0-5.0) × 10⁶ CD34⁺ cells containing more than 1.010⁶ per kg CD34⁺CD45^low GSK. Presence of CD34⁺CD45^- HSC subpopulations in the amount of 15% or more of the total HSC pool will provide the recovery of neutrophils within 10 days, see Table 4.

The amount of myeloid-restricted CD33 ⁺ HSCs in BMCP "HemaComb" is significant, the smaller number of which in the graft is associated with shorter recovery times for neutrophils. The reduction in critical thrombocytopenia to 10 days is due to the presence of more than 35.0% of CD34 ⁺ Thy-1 ⁺ cells and more than 5.0% of CD34 ⁺ CD7 ⁺ HSCs among the transplanted HSCs.

Thus, the use of BMCP "GemaComb" for the treatment of cancer patients can be considered as a method of choice for the treatment of complications after chemotherapy, radiation therapy, combined with rehabilitation therapy. The proposed method of treatment of BMCP "GemaComb" today is a fairly effective and safe way of treating cancer patients, which can lead to a real improvement not only in their condition, but also in social rehabilitation in society. The higher efficiency of the use of BMPK "HemaComb" in the rehabilitation of cancer patients after radiation and chemotherapy, as well as the use in weakened and gerontological patients has been shown. The economic efficiency of the use of BMCP "HemaComb" in patients in comparison only with autologous HSCs in the LA of mobilized bone marrow MNCs is obvious and does not cause doubts. Returning patients to normal social and social activities, rehabilitation of cancer patients after radiation and chemotherapy, improving the quality of life of weakened and gerontological patients, even with a slight improvement in their quality of life, carries a great economic feasibility and social justification, both taking into account the resumption of labor activity, so and taking into account the reduction in costs for long-term and ineffective pharmacological treatment and only physical rehabilitation.

The effectiveness of the drug "GemaKomb" in neurological diseases

The analysis of the effectiveness of the drug "GemaKomb" is based on a clinical study and a retrospective long-term study of 2780 case histories of 458 patients with various organic diseases of the central nervous system, who underwent long-term inpatient experimental treatment at the clinic of restorative interventional neurology and therapy "NeuroVita" (Russia, Moscow) since September 2002 year to March 2016 within the framework of the branch program of the Russian Academy of Medical Sciences "New cell technologies for medicine" (program manager, academician of the Russian Academy of Medical Sciences, prof. VK Yarygin, research coordinator, prof. AS Bryukhovetsky). The distribution of patients by the nosology of nervous diseases is presented in Table 5.

The distribution of patients by age and sex and the area of spinal cord injury is presented in Table 6.

As can be seen from the presented materials, patients of different age groups took part in the clinical study. The researchers tried to ensure that the control groups correlated in age with the experimental group. It so happened that the control group included only one men, but the gender feature practically did not affect the results of the study.

The analysis of the application of various strategies for the use of SCs and technologies of regenerative medicine in patients with various neurological diseases was carried out. The distribution of patients depending on the strategy of applying regenerative medicine technologies (conservative, surgical and combined) are presented in Table 7. As can be seen from Table 7, the main strategy for the clinical use of SC was the strategy of cell therapy, which amounted to 72.4%, tissue engineering in an isolated form was used only in 13.8%, and in 13.7% of cases it was investigated to combine and supplement tissue engineering with cell therapy.

Patients with various neurological diseases included in the program were under constant dynamic observation and studied for 3-8 years, undergoing control inpatient examinations every 3 months with a mandatory clinical examination, including neurological examination, MRI of the brain (GM) or spinal cord (SM) with contrast enhancement (in 50 patients with traumatic SM disease, tractography of the pathways of GM and CM based on diffuse tensor studies was performed), neurophysiological examination in the form of electroneuromyography, electroneuromyography with somatosensory evoked potentials, test studies on different scales ( ASIA scale, FIM scale), X-ray control of the knee and hip joints in accordance with the existing research protocol approved by the Academic Council and Ethics Committee of the Russian State Medical University named after N.I. Pirogov (Russian State Medical University). All interim and final reports on these studies were submitted annually to the administration of the Russian State Medical University and the Ministry of Health of the Russian Federation. For the treatment of different types of damage to the nervous tissue of GM and SM, various types of cell preparations were used: a preparation of HSCs and HKPs mobilized from the bone marrow (CD34 +, CD45-) (the manufacturer of the preparation is the Blokhin Russian Cancer Research Center), a preparation of neural SC ( CD 133+), isolated from the patient's olfactory lining (manufacturer of the drug - V.P.Serbsky State Scientific Center for Social and Forensic Psychiatry) and MSSK, isolated from the patient's bone marrow (manufacturer of the drug - Federal Research and Clinical Center of FMBA of Russia) ... All cellular material was standardized and certified by government research institutions, and government quality certificates for the material were pasted into patient records.

Evaluation of the effectiveness of cell therapy largely depended on the nosological affiliation of the disease and the goals of the SC application. An analysis of the results of effectiveness in various nosologies of diseases of the central nervous system is presented in tables 8, 9.

According to the results of early studies by the authors, the effectiveness of the use of SC preparations in the mode of cell therapy is the most effective and ranges from 35 to 56% (Bryukhovetsky A.S., 2003). This study shows that therapy is effective in 55.4% of cases, and highly effective cell therapy was noted in 15.1% of cases. The effectiveness of tissue engineering and bioengineering is about 41-49.6%. So far, it has not been possible to increase the efficiency of regenerative medicine technologies above 56%, despite the variety of uses of various cell preparations. At the same time, the use of cell preparations turned out to be quite safe.

List of abbreviations used

PE - phycoerythrin label

PerCP - Peridinine Chlorophyll Tag

ALS - amyotrophic lateral sclerosis

BMCP - biomedical cell product

HKP - hematopoietic progenitor cells

G-CSF - granulocyte colony-stimulating factor

GM - brain

HSC - hematopoietic stem cells

DMSO - dimethyl sulfoxide

KP - progenitor cells

CSF - colony stimulating factor

LC - leucoconcentrate

MGB - gliobastoma multiforme

MCA - monoclonal antibodies

MNC - mononuclear cells

MSSK - mesenchymal stromal stem cells

MSCP - mesenchymal stromal progenitor cells

NHL - Non-Hodgkin Lymphomas

OK - tumor cells

AML - Acute Myeloid Leukemia

PC - peripheral blood

POV - damaged cells

RIF - reaction of immunofluorescence

RNA - ribonucleic acid

RSC - cancer stem cells

SC - stem cells

CM - spinal cord

CNS - Central Nervous System.

Claims (31)

1. Biomedical cell product (BMCP) for the treatment of oncological or neurological diseases, containing a line of autologous or immunocompatible allogeneic mesenchymal stromal stem cells (MSSK) with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and mesenchymal stromal progenitor cells (MSCPs) with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 +, isolated from native bone marrow or adipose tissue and cultured in vitro in a medium , contributing to mesenchymal differentiation (component A), unmanipulated autologous or allogeneic mobilized mononuclear cells (MNC) isolated from peripheral blood leuconcentrate (PC PC), purified from blood plasma, erythrocytes and granulocytes (component B), and an auxiliary substance in the form of a sterile transfusion environment (component B) with the following ratio of components (vol.% of the total amount of BMCP): component A - 1-19.5; component B - 79.5-90; component B - the rest. 2. BMCP according to claim 1, characterized in that component B contains αβ T cells, γδ T cells, NK and NKT cells, B1 and B2 cells, hematopoietic stem cells (HSC) with markers CD34 +, CD45 + , HLA DR +, hematopoietic progenitor cells (HCP) with markers CD34 +, CD45-, HLA DR-, MSSK with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and MSCP with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + with the following ratio of components (vol.% of the total amount of component B): αβ T cells - 55-60; γδ T cells - 10-15; NK and NKT cells - 15-20; B1 and B2 cells - 10-15; GSK and GKP - 1.5-2.5; ISSK and ISKP - 1.5-2.5. 3. BMCP according to claim 1, characterized in that component B contains αβ T cells, γδ T cells, NK and NKT cells, B1 and B2 cells, hematopoietic stem cells (HSC) with markers CD34 +, CD45 + , HLA DR +, hematopoietic progenitor cells (HCP) with markers CD34 +, CD45-, HLA DR-, MSSK with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and MSCP with markers CD10 +, CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 +, and component B is partially purified from αβ T cells at the following ratio of components (vol.% of the total amount of component B): αβ T cells - 30-55; γδ T cells - 15-20; NK and NKT cells, 17-30; B1 and B2 cells - 10-15; GSK and GKP - 1.5-2.5; ISSK and ISKP - 1.5-2.5. 4. BMKP according to one of paragraphs. 1-3, characterized in that 0.9% saline NaCl with granulocyte colony-stimulating factor (G-CSF) in a volume of 5-100 vol.% Is used as an auxiliary substance. from the volume of physiological NaCl solution. 5. BMPK according to one of paragraphs. 1-3, characterized in that a biodegradable polymer matrix with granulocyte colony-stimulating factor (G-CSF) transfused in it in a volume of 5-20 vol.% Of the matrix volume is used as an auxiliary substance. 6. BMPK according to claim 1, characterized in that the alogenic MNCs are immunocompatible with respect to the blood group, Rh factor and blood immunophenotype. 7. A method of obtaining ex tempore biomedical cell product according to any one of paragraphs. 1-6, which includes: obtaining a line of autologous or immunocompatible allogeneic mesenchymal stromal stem cells (MSSC) with markers CD10 +, CD13 +, CD44 +, CD90 + (Thy-1), CD105 +, CD34-, CD45- and CD117- and mesenchymal stromal progenitor cells (MSSC) with markers CD10 , CD13 +, CD44-, CD90 + (Thy-1), CD105 +, CD34 +, CD45 + and CD117 + by isolating these cells from native bone marrow or adipose tissue and further cultivation in vitro in an environment promoting mesenchymal differentiation (component A), obtaining unmanipulated autologous or allogeneic mobilized mononuclear cells (MNCs) by isolation from peripheral blood leuconcentrate (PC PC) with subsequent purification from blood plasma, erythrocytes and granulocytes (component B) and ex tempore mixing of components A, B and an auxiliary substance in the form of a sterile transfusion medium (component C) with the following ratio of components (vol.% of the total amount of BMCP): component A - 1-19.5; component B - 79.5-90; component B - the rest. 8. The method according to claim. 7, characterized in that the obtained components A and B are cryopreserved, and before mixing them ex tempore with component C, they are thawed. 9. The use of a biomedical cell product according to any one of paragraphs. 1-6 for the treatment of cancer. 10. The use of a biomedical cell product according to any one of paragraphs. 1-6 for the treatment of neurological diseases.

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