5 Technical Field

Technical solution relates to the umbilical cord blood collection processing, processing and preservation of collected umbilical cord blood and allogeneic use of cord blood for processing and production of advanced therapies intended for clinical evaluation and subsequent treatment of patients with selected diseases.

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Background of the art

Over 80% of deaths worldwide are caused due to disease or health condition incompatible with human life. Almost 30 % of this is caused by cardiovascular diseases taking the first place. At the second place are deaths caused by infectious

L5 diseases (23%), the third place is occupied by ischemic heart disease (13 %).

Cancer is in fourth place (12 %), stroke in fifth place (10 %). In the United States, where there are accurate statistics of diseases and treatment methods, the number of patients suffering from one or more types of cardiovascular diseases is 82.6 mil. (AHA / American Heart Association, 2012), i.e. 26 % of the population. Of that

>o coronary heart disease accounts for 16.3 mil. (5% of the population). According to the AHA, in 2030 over 40 % of the U.S. population will suffer from cardiovascular disease. Almost 7 % of the population suffer from diabetes, i.e. 21.1 million people (CDC / Centers for Disease Control and Prevention) and 68 thousand of amputations are performed each year due to the consequences of the disease. In 2012, the

>5 American Cancer Society expects to record 1.6 million newly diagnosed cancer cases and 577 thousand deaths caused by cancer. 50 million people suffer from some type of athritis (16 % of the population) and by 2030 this number is expected to increase to 67 million. Unhealthy lifestyles and advancing age leads to increasing frequency of occurrence of these diseases and as is evident from the previous

50 paragraph, these diseases are a very frequent cause of death. In addition to treatment of chronic disease, there is also a broad range of treatments that focus on acute and traumatic conditions. Their frequency is significantly lower than that of the above-mentioned diseases, but often they often affect people of working age and also children. Fast and effective treatment is therefore necessary, as it allows to restore a healthy individual back to a full life and work. Currently, the treatment of the above diseases and conditions is based on conventional drugs and synthetic substitutes, which are combined with modern surgical techniques. Furthermore, there are advances in the application of new drugs, the production of which is based on biotechnological processes. Historically the youngest is the area called Advanced cellular therapies. Here, the first preclinical results were recorded in 1990. This new area of medicine still does not have a large number of clinical outcomes compared to conventional methods of treatment. However, the existing results of Advanced therapies significantly exceed those results that are achieved by conventional medicine. Case reports and clinical tests confirmed the possibility of treatment of so far untreated conditions.

The area of Advanced Therapies aims to treat acute and chronic conditions of humans or tissue replacement. A major long-term scientific goal is to extend the life and function of human organs, to treat cancer and chronic or degenerative diseases. The objective thus involves a gradual extension of human life utilizing cells. Treatment based on one ^' s own (autologous) or foreign (allogeneic) cells and supporting regeneration products in the treatment of trauma, developmental defects, cancer resections and musculoskeletal diseases is based on the availability of new functional biomaterials and their clinically relevant forms. The most promising group of future biomaterials for tissue regeneration, replacement of organs are the synthetic biocompatible polymers (PLA, PGA, PEG, PHB, etc.), natural polymers (type I collagen, fibrin, chitosan, cellulose, starch, HA, etc.) and their hierarchical, functional composites with resorbable layered nanoparticles. The use of synthetic polymers with the ability to manage their molecular structure and morphology of cellular carriers directly through manufacturing technology is subject to achievement of biocompatibility in clinical use, and to minimization of the impact of the products of biodegradation on the living cells (pH, C0 _2> etc.). Although these problems are eliminated with natural polymers, there is a major problem involving the absence of ^' technology that would enable reproducible manufacturing of carriers made of natural polymers and the poor mechanical properties of such carriers. Nano-fiber carriers for cell therapy started to be developed in the mid- 1990s using technique called electrospinning, and at that time nanofibers from most of the known biomaterials were successfully produced. The biggest motivation for rapid take off of this development was probably the morphological similarity of the natural extracellular matrix (ECM, Extra Cellular Matrix) and nanofiber media produced by electrospinning. ECM must mimic each carrier of tissue engineering. The carrier provides the cells with temporary mechanical support, it is sufficiently "chemically attractive" and its toxicity should be minimal, while it should decompose after a defined time. Applications for nanofibers include wound dressing, burns, artificial skin, cartilage, bone, tendons, nerves etc. The cell carriers have not just passive support role of growth vectors and mechanical protection of cells in the growth process, but their mechanical properties determine the ease of surgical handling, as well as - in conjunction with controlled adhesion of cells to the carrier - the proliferation and differentiation of the implanted cells and their morphology secreted into the ECM, and thus the entire tissue / organs. In addition to biomechanical stimuli, the regeneration and growth of new tissue require significant spatial and temporal biochemical stimulators (TGF, BMP, cytokines, chemokines, etc.), the controlled release of which into the cellular carriers in a biologically active form and in sufficient quantity has not yet been achieved and angiogenic factors stimulating the tissue vascularization necessary for the survival, growth and differentiation of cells 30 in the 3D carriers.

Equally important is the identification of the most appropriate sources of regenerative cells (autologous, allogeneic, MSM, PLSC, DPSC, mature embryo, etc.) or their combination for a given clinical situation. In vitro tissue growth with subsequent implantation is promoted especially in poorly regenerating tissues (cartilage, tendon, dentin) or large segments of tissue, whereas in case of well-healing tissue (bone) and in small defects in vivo direct implantation of carriers with cell cultures or without them is preferred. In vitro procedure is burdened by more legislative barriers than in vivo. Lack of proven practices of rapid generation of vascular systems that would provide nutrition and respiration of cells in larger 3D carriers still limits their use to thin planar structure. The greatest challenges of field include the development of a reproducible method of preparing large vaskularizable 3D implants, combination of cell types and components of carriers with controlled release of drugs and biochemical stimulants.

Cord blood is the blood contained in the umbilical cord and placenta. Umbilical cord blood is a source of various types of stem cells, in particular the hematopoietic stem cells and progenitor cells, as well as mesenchymal stem cells and other somatic stem cells.

Umbilical cord blood is harvested during childbirth, and further processed. It can be stored frozen for several years, and used for allogeneic and autologous transplantation.

The widest use is in allogeneic transplantation. They are used particularly for diseases such as congenital types of leukemia.

Autologous transplantation is performed in conditions that require recovery of hematopoiesis such as malignant aplastic anemia or to restore hematopoiesis after chemotherapy. This use is still limited by the fact that it is a relatively new method, where the number of potential beneficiaries, i.e. those who have stored their own autologous umbilical cord blood is low compared to the incidence of the disease in this age group.

The advantage of the umbilical cord blood is the fact that it is collected at the beginning of life, when the stem cells are still unburdened by acute disease.

Stem cells from cord blood may also be applied in the context of regenerative medicine and cell therapy, where a number of experiments and clinical trials are ongoing.

Umbilical cord blood is harvested during childbirth, from umbilical cord vessels and in case of combined collection after delivery of the placenta also from the surface of placental blood vessels. Blood is placed in a sampling set and is further processed. An essential criterion for further use of allogeneic transplant is the volume of the collected blood, and the total content of the nucleated cells.

Adequate volume of collected blood is more than 100 ml, and the number of nucleated cells (TNC) of more than 150 x 10 ⁷ cells, and also a sufficient amount of CD34+ cells to provide good graft survival, wherein a higher than normal amount reduces rejection by the recipient organism (GVHD, graft versus host disease).

Umbilical cord blood, which does not meet the criteria for allogeneic transplantation or donor transplantation, is currently mostly disposed of, and thus the source of quality stem cells is wasted.

The present invention addresses the issue of the umbilical cord blood collections that could not be used until now - i.e. those which do not meet any of the criteria for transplant, describing the method of their further processing and production of products for clinical trials and additional patient treatment in case of certain diseases.

Summary of the invention

The present invention relates to a method of processing of collected samples of umbilical cord blood (UCB), wherein a) umbilical cord blood is subjected to at least one quality test ; b) umbilical cord blood that meets the criteria for at least one of the quality tests in a) is treated as an autologous or donor umbilical cord blood respectively; c) umbilical cord blood that does not meet the quality criteria of b) for autologous or donor umbilical cord blood is processed as waste blood.

The quality test as per a) is preferably selected from the group of tests determining the number of nucleated cells TNC; determination the amount of CD34+ cells; determination the volume of umbilical cord blood; determination of HLA phenotype of blood grouping and Rh factor; determination of blood cell count and vitality; serological blood tests; and blood cultures.

In a preferred embodiment, the quality test used in a) is selected from the group of: the number of nucleated cells TNC ; the amount of CD34+ cells; and determination of the volume of umbilical cord blood. Unlike the use of umbilical cord blood for transplantation, the specific further use does not need to meet all quality tests for transplantation, for example the corresponding HLA phenotype, the same blood group and Rh factor.

Other specific uses of waste blood, however, may require compliance with one or 5 more additional criteria different from the criteria, whose fulfillment leads to classification of the blood as an autologous blood, or donor umbilical cord blood.

Tests on the additional criteria may be performed simultaneously with the tests as per a) above, which is advantageous in terms of blood processing speed or using blood from paragraph c) falling under the category of waste blood, which is o advantageous in terms of cost savings for unused tests, or a combination of both approaches. Due to the high costs of tests this decision is very important.

In another embodiment, the waste blood as per c) is further classified based on the fulfillment of the criteria of at least one quality test used in a) different from the quality tests as used in b) for autologous or donor umbilical cord blood. 5 In yet another embodiment, the waste blood from point c) is further classified in d) on the basis of meeting the criteria of at least one additional quality test different from the quality tests as per point a).

In one embodiment the quality test used in d) is selected from the group: determination of the number of nucleated cells TNC; determination of the amount of o CD34+ cells; determination of the volume of cord blood, the determination of HLA phenotype; blood-grouping and Rh factor; determination of the blood counts and vitality; serology blood tests; and blood cultures.

Another important embodiment of the invention relates to a method of processing of umbilical cord blood collections as described above, whereas additional fractionation 5 of waste blood is performed.

Fractionation is preferably carried out by methods known in the field, e.g. by various modifications of centrifugation such as gradient centrifugation and in another embodiment a corresponding method of fractionation to obtain mesenchymal stem cells is used. A very important aspect of the present invention is the possibility of mixing individual samples of umbilical cord blood. The condition for such successful mixing waste blood may the compliance with one or more additional criteria different from the criteria the fulfillment of which results in the classification as autologous blood or 5 donor umbilical cord blood, and may allow the use of even small amounts of cord blood unsuitable for transplant.

Another embodiment of a method of umbilical cord blood processing described herein thus relates to a process, which involves further mixing of waste blood or its fraction from at least two umbilical cord blood collections.

L0 For many important applications such as wound dressings, the waste blood or its fractions are further bound to a carrier.

Carriers are preferably based on nanofibers, nanocarriers, collagen carriers, hydrogels, textile meshes, tissue matrix and their combinations, particularly of nanowires mounted on a grid of biocompatible material.

L5 Waste blood and/or its fractions, alternatively in a form bound to a carrier, possibly mixed from several donations, obtained as described above is suitable for the manufacture of medicinal products for cardiology and neurology, in particular for the treatment of Parkinson's disease, post-traumatic and ischemic brain and spinal cord lesions, the treatment of autism and hearing loss in children.

^» o Alternative uses involve dressing of superficial wounds in major surgery and supportive indications in oncology.

Waste blood and/or its fractions, alternatively in a form bound to a carrier, possibly mixed from several collections, obtained as described above are also suitable for example as a source of induced pluripotent stem cells.

»5 Waste UCB may be stored in several ways: i) individually by fractions

ii) entire graft

iii) only selected separated fractions. In practice, the following materials are stored:

i) the umbilical cord as a whole,

ii) individual vessels, Wharton's jelly, the umbilical cord wrap and collected umbilical cord blood,

iii) specific cells or^mesenchymal stem cells extracted from the above components.

Different ways of storage means different use of the UCB and different products.

Priority areas for the use of waste blood according to the invention in the field of modern therapy are especially: a) the healing of wounds, burns, skin (including deep third degree burns with skin loss in entire thickness), replacement and augmentation of soft tissues (reconstructive and plastic surgery); treatment of musculoskeletal system; cord blood bank, b) control of diabetes and treatment of the consequences of diabetes, cardiology, stroke; c) oncology and neurology.

Nanocarriers, collagen carriers, hydrogels, textile gauze, tissue matrix and the combination of the above can be used as carriers for therapeutic use of the waste blood.

Therapeutic use of stem cells from the waste blood with carrier is particularly applied in the healing wounds and burns, to cover superficial wounds and in major surgery, cardiology and neurology. In neurology, it is primarily used for neural damage, Parkinson's disease, brain and spinal cord lesions - traumatic and ischemic, but also in the treatment of autism and hearing loss in children.

Therapeutic use without the use of a carrier for example involves autoimmune diabetes (diabetes mellitus) type I and II, amyotrophic lateral sclerosis, as well as alopecia (hair loss), and psoriasis.

Stem cells derived from waste UCB can be used in a number of supporting indications in oncology.

Waste UCB can be used as a source of induced pluripotent stem cells.

Examples

General principles of umbilical cord blood processing

Collection of UCB, or modification of the volume (see below) and other operations are carried out in a closed system, dimethyl sulfoxide is added through a bacteriological filter to prepare cryo-preservation solution. Processing of samples for freezing takes place in a laminar flow of air purity class A in compliance with the principles of aseptic procedures.

If necessary the processing of the umbilical cord blood my further involve blood fractionation using methods such as centrifugation.

Sampling and testing

Samples are collected and one or more of the following tests and investigations known in the field are performed:

- number of nucleated cells TNC

- amount of CD34+ cells

- determination of the volume of cord blood

- determination of HLA phenotype

- blood group and Rh factor

- blood count and vitality by trypan blue staining

- serological tests of mother ^' s blood

- blood culture

Separation of waste blood based on the criterion of the number of TNC cells

If the number of cells TNC is higher than 150 x 10 ⁷ cells, blood is used for further processing as waste blood.

Separation of waste blood based on the criterion of the number of CD34+ cells

If the collected cord blood does not have sufficient amount of CD34+ cells to ensure a good graft survival, the blood is used for further processing as waste blood.

Separation of waste blood volume based on the volume of collected blood

If the volume of cord blood collected with the anticoagulant solution is 100 ml to 134 ml, this amount of suspension can be processed directly, without pre-treatment of volume. Correction of the excessive amount of blood is performed by removing excess plasma If the volume of cord blood collected with an anticoagulant solution is less than 100 ml, it is not recommended to store this blood. Such blood is used for further processing as waste blood. Separation of waste blood based on the failure to comply with other criteria

If one of the selected criteria from the above listed is not complied with or other criteria or a combination thereof, the blood is used for further processing as waste blood. Disposal of umbilical cord blood

In case of positive serology of the umbilical cord blood for HBsAg or HIV, the blood is disposed of after processing and freezing.

Cryo-preservation

Umbilical cord blood (= cellular suspension) is mixed with cryoprotective solution and frozen at a controlled temperature of -175 ° C. This procedure retains the viability of cells in suspension.

The cryoprotective solution used is DMSO with final concentration of 10%. The initial material is 100% DMSO solution supplied in vials with piercing stopper with volume of 10 ml. This concentrate is used to prepare a 30% solution of DMSO in 5% albumin, and this is then mixed with freezing suspensions at the ratio of 2/3 suspension, 1/3 of DMSO (30 %), which provide a final concentration of 10% DMSO in a cryo suspension. DMSO is toxic for cells and toxicity increases with increasing temperature, therefore mixing of DMSO with the cell suspension is carried out at a temperature of 0-4 ° C. Immediately after this the mixture is frozen.

Mixing of cord blood

6 umbilical cord blood samples were mixed: 20 ml, 33 ml, 51 ml and 89 ml, that did not meet the criterion for the volume of umbilical cord blood, but did meet the criteria of cell vitality test by trypan blue staining and were negative for antibodies for hepatitis, HIV and syphilis serology based on blood test using e.g. RR test and TPHA test. Blood stem cells were isolated from the mixed blood by gradient centrifugation and the isolate was directly used for attaching to the nanofiber mesh.

Therapeutic use with carrier

Mesenchymal stem cells from the UCB according to the invention were used on a layer of nanofibres for dressing of superficial wounds. For this purpose, a mesh of biocompatible material was used, with the inner space filled with a layer of nanofibres. Monofilament warp knit of colorless filament was used as a matrix and spinnabie polymer based gelatin was used as a layer of biocompatible polymer nanofibres.

This layer contained a growth factor as the active ingredient for stimulating the cultivation, and antiseptic to prevent entry of undesirable microorganisms.

Prior to the application of mesenchymal stem cells, the medium was sterilized by gamma radiation. Mesenchymal stem cells from waste umbilical cord blood according to the invention were then placed on a sterilized nanocarrier by pipette in the form of a suspension in nutritional solution.

After storing the cells adhered to the polymeric nanofibers and in the course of a five day cultivation they created more layers. Culture medium was changed once a day. After culturing the nanocarrier was removed from the culture medium and the excess of the medium was removed from by dripping.

Industrial application

The method of processing and preservation of waste umbilical cord blood according to the solution can be used for processing and preservation of waste umbilical cord blood and for allogeneic cord blood processing and production of advanced therapies intended for clinical trials and additional patient treatment for certain diseases.

Priority areas of interest in the field of advanced therapies are: a) the healing of wounds, burns, skin (including deep third degree burns with full thickness skin loss), replacement and augmentation of soft tissue (reconstructive and plastic surgery) treatment of the locomotor system; cord blood bank, b) diabetes control and treatment of the consequences of diabetes, cardiology, stroke, c) oncology and neurology.