TW200916583A - Cell expansion - Google Patents

Abstract

The present invention provides in one aspect a method of ex-vivo expanding umbilical cord blood CD34+ progenitor cells, comprising: (a) encapsulating umbilical cord blood cells in a support matrix; (b) seeding the encapsulated cells into a dynamic culture vessel; and (c) culturing the cells in the dynamic culture vessel under conditions allowing for CD34+ progenitor cell expansion.

Description

200916583 IX. INSTRUCTIONS: FIELD OF THE INVENTION The present invention relates to the field of methods for amplifying cell populations, particularly umbilical cord jk stem and/or progenitor cells. 5 [Prior Art] Cord blood stem cells have been transplanted more than 8,000 times since the treatment of 45 different blood disorders since 1988 and have been used to repair and restore bone marrow after high-dose chemotherapy for cancer. However, the use of cord blood units as a source of stem cells for transplantation is limited because of the relatively low number of CD34+ progenitor cells in a single cord blood unit. An average cord blood unit contains only enough CD34+ progenitor cells to treat a patient weighing 30 kg •. The mixing of the umbilical cord also presents some problems. And it is not considered to be the ideal standard. There is therefore a clear need for CD34+ cell expansion 15 technology to enable cord blood units to be used to treat all categories of patients in need (Kurtzberg ei α/. 1996; : Wagner et al. 1996; Kapelushnik et al) 1998; Shpall et al. 2000; Jaroscak ei a/. 2003a) ° In vitro expansion of CD34+ progenitor cells over the years has been attempted in a variety of static and dynamic systems, including the use of bioreactors. . These studies have demonstrated the potential for cell expansion but at the same time have not led to routine clinical applications. Continued understanding, discovery, and isolation and identification of cytokines, cell subtypes, and the development of various bioreactors and support scaffold concepts 200916583 continue to drive future amplification technologies for CD34+ progenitor cells. Some clinical trials have been planned but the technology still needs further improvement (Hoffman et al. 1993; Wagner 1993; Andrews et al. 1994; Purdy et al. 1995; Gehling et al. 1997; Bachier et al. 1999; 5 Chabannon Et al. 1999a; McNiece et al. 1999; Nielsen 1999; McNiece et al. 2000a; McNiece and Briddell 2001; Noll et al. 2002; Wolf 2002; Knustsen et al. 1998; Vilquin ei a/. 2002).

Lui et al. (2006a) have previously attempted to amplify umbilical cord blood cells encapsulated in calcium alginate using exogenous cytokines and rabbit mesenchymal stem cells in static culture. This method was found to have a negative impact on the total number of CD34+ cells and CFU-GM. This is important because the amplification with loss of multipotency is not very practical. 15 Subsequent work by Lui et al. (2006b) uses a rotating wall vessel bioreactor constructed for a specific purpose and a cell with exogenous cytokines but no cell coating. Steps. WO 2005/007799 describes a method for the expansion of stem/source progenitor cells in vitro in a bioreactor. A wide range of cell types are discussed, such as skin cells, hepatocytes, nerve cells, and bone cells. A variety of different conditions have been employed, including the use of exogenous cytokines and transition metal (note copper) chelating agents. Transition metal chelators are used to inhibit differentiation of progenitor cells. However, this document does not specifically teach the use of a coating to amplify CD34+ progenitor cells in a 200916583 dynamic culture vessel. US6440734 teaches methods and devices for the long-term culture of hematopoietic progenitor cells. This document describes the expansion of cord blood 5 stem cells in a static, button-coated scaffold without the use of exogenous cytokines but in the presence of serum. It is suggested to use a coating or impregnation of a range of biologic agents (including 'gels'). WO 2006/081435 teaches a method of providing readily available cellular material derived from cord blood and its compositions. This document describes the novel use of electromagnetic 10 radiation combined bioreactor technology. The coating is not mentioned in this technique. W02006/079854 teaches a method for embryonic stem cell culture, wherein • coated embryonic stem cells are maintained and differentiated in 3D culture. However, this document does not provide a description of cell types (such as cord blood 15 stem or progenitor cells, including CD34+ progenitor cells derived therefrom), as its teachings are limited to embryonic stem cells. There are thus some existing methods for amplifying specific types of stem and source progenitor cells. However, there is a clear need for improved methods for growing larger numbers of stem and progenitor cells (especially CD34+ progenitor cells derived from cord blood) for clinical transplantation applications and for regenerative medicine. SUMMARY OF THE INVENTION Accordingly, in one embodiment, the present invention provides a method for in vitro expansion of mitochondrial blood CD34+ progenitor cells, comprising: (1) coating a blood cell of 200916583 cells on a support matrix (support) (b) planting the coated cells into a dynamic culture vessel; and (c) culturing the cells in the dynamic culture vessel under conditions that permit expansion of the CD34+ progenitor cells. In another embodiment, the invention provides a coated cell, or an expanded population, of cord blood CD34+ progenitor cells obtainable or obtained by the method defined by the above. In still another embodiment, the present invention provides a dynamic culture vessel comprising cord blood cells coated in a support matrix. In still another embodiment, the invention provides a coated cell, such as a cord blood CD3 4 + progenitor cell obtainable or a method obtained by the method defined above, or an expanded population for use in Regenerative therapy. In still another embodiment, the invention provides a coated cell, or an expanded population, for regenerative, for example, or obtained from a cord blood CD34+ progenitor cell obtained by the method defined above. 15 uses of therapy. In still another embodiment, the present invention provides a coated cell of a cord blood CD34+ progenitor cell obtained by the method defined by the above I face, or an expanded population for preparation. The use of a medical agent that treats a disease or condition requiring regenerative therapy. In yet another embodiment, the invention provides a method for treating an individual in need of regenerative therapy, comprising administering to the individual a therapeutically effective amount of one of the source ancestors obtained by the method as defined above cell. In still another embodiment, the invention provides a method comprising: (a) 200916583 coating cord blood cells in a support matrix (eg, a suitable protective support matrix); and (b) freezing the coated cells . The cells used in the method of the present invention can be obtained from any species of an animal having an umbilical cord (i.e., from any placental mammal). In particular, the cells may be of human, non-human primate, horse, dog, cow, pig, goat, sheep, Confucian or murine source. In a preferred embodiment, the cells are umbilical cords derived from an animal of a species, the species being the equine, especially the genus, such as a horse, zebra or scorpion; optimally such cells From one horse. In another preferred embodiment 10, the cells are human cells, i.e., derived from human umbilical cord blood. Preferably, the cells are coated within 72 hours of collecting cord blood. Since the collection of cord blood typically occurs after delivery, the cells are preferably coated within 72 hours postpartum, more preferably within 48 or 24 hours after birth. Alternatively, the umbilical cord can be frozen and the 15 cells can be collected and coated after thawing. The support matrix preferably comprises a hydrogel. Preferred materials for use in the support matrix include alginic acid, fibronectin, and mercaptocellulose, including each of those materials, or mixtures thereof. The support matrix is preferably in the form of a bead. Hydrogels exhibit lower mass transport problems than 20 scaffolds. The hydrogel can also be dissolved to allow easy recovery of the cells. In one embodiment, the cells are coated in a bead, such as a hydrogel bead, at a density of from 100 to 300,000 cells per bead, for example from 500 to 300,000, 3000 to 300,000, 3000. To 200916583 200,000, 5000 to 200,000, 500 to 10 〇, 〇〇〇, about 5,000 or about 200,000 cells per bead. The beads may have an average diameter of 0.1 to 5 mm, 0.5 to 5 mm, i to 5 mm, 2 to 3 mm or, in a specific embodiment, about 2.5 mm. 5 After coating the cells in a support matrix, in one embodiment, the coated cells can be frozen. After freezing, the cold-coated coated cells can be stored indefinitely for later use. When desired, the frozen coated cells can be thawed prior to other methods being performed, i.e., seeding 10 or culturing the thawed coated cells in the dynamic culture vessel. These examples enable umbilical cord blood expansion to be performed directly from freezing and thus limit post-thaw operation and subsequent cell loss and injury prior to expansion. The method is preferably carried out in the absence of serum, for example, in a specific example, the cells are cultured in a serum-free medium. Preferably, the cells can also be cultured in the absence of a transition metal chelating agent. In the specific example, the dynamic culture vessel is a bioreactor. Even more so, the biological benefit is the rotary wall container bioreactor. A preferred dynamic culture vessel for use in the present invention is a harv bio 2 reaction or a NovaPodTM bioreactor. The cells, such as children, can be cultured in a medium free of cytokines' or alternatively in a medium containing one or more cytokines. When the medium contains one or more cytokines, in one embodiment, the cytokines are early acting cytokines. Examples of early acting cytokines include, but are not limited to, stem cell factors, FLT3 ligand, interleukin-6 and interleukin-3. In another embodiment, the cytokines comprise one or more stem cell factors (SCF), jk small plateogen (thromppoietin, TPO), and granulocyte-colony stimulating factor , G-CSF), for example, the medium may contain SCF, TPO, and G-CSF. For example, the cytokine can be one to one, one to five, one to two hundred, one to one, one to one, one to fifty or one to! The concentration of 〇 ng/ml is present. 10 In one embodiment, the cells are cultured in the presence of mesenchymal stem cells. The mesenchymal stem cells may be coated with or contained in the cord blood cells. The coated cells are preferably cultured in a suspension culture in step (c). According to a particular embodiment of the invention, an expanded population of cells obtained from the practice of the invention (derived from a single cord blood sample) can be administered to a single subject in need of regenerative therapy. In other words, the source progenitor cells found in a cord blood unit taken from a single newborn can be expanded enough to provide sufficient cells to treat a single patient, such as an adult patient. The expanded population of cells can be used to treat any disease or condition requiring regenerative treatment, particularly a disease or condition that is treated with cells of the myeloid lineage. Applications of expanded cell populations include, but are not limited to, bone marrow transplantation, transfusion medicine, organ weight, regenerative medicine, tissue engineering, gene therapy, toxicology, and research. Diseases to be treated, particularly preferably according to embodiments of the invention 200916583 include blood disorders and bone marrow loss following chemotherapy. The invention can also be used for screening as well as for toxicological studies. Specific embodiments of the invention may also advantageously facilitate expansion of the cord blood cell population to make them more useful in therapy. In particular, the use of a coating combination in a 5 holding matrix allows for the reconstitution of an environment in vitro in a dynamic culture vessel, which is particularly suitable for the expansion of blood CD34+ progenitor cells and inhibits cell differentiation. Moreover, the methods of the present invention do not require the use of reagents (such as serum or transition metal chelators), the presence of which may be problematic for downstream applications, such as human cell transplantation therapies. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods for in vitro expansion and culture of ancestral cord blood stem cells and stem cells and their possible uses. In particular, in a specific example, human wilted blood CD34+ progenitor cells (as in, for example, in the chromophore layer) can be extensively expanded and grown in vitro in the bioreactor according to the method of the present invention. . In particular, in one embodiment, the method can be used for large-scale in vitro expansion of stem and source progenitor cells, resulting in a regenerative population of large numbers of stem and/or progenitor cells, which can be applied, for example, for example Bone marrow transplantation, transfusion medicine, organ reconstruction, regenerative medicine, tissue engineering, gene therapy, toxicology, and research. Attention is drawn to the fact that a specific example of the method is stroma-cell free. These specific examples also do not require the passage of cells. In certain embodiments, the method can involve, for example, a 2-100 fold, such as a 2-50 fold, a 2-20 fold, or a 5-20 fold umbilical cord blood CD34+ progenitor cell within 2 to 30 days. Amplification, preferably 12 200916583 5-1 fold amplification in approximately 10 days. In order to provide sufficient cells for transplantation, an average cord blood unit is 3χ1 〇8 and total nucleated cells must be expanded 4-5 times. However, a key parameter is that total CD34+ progenitor cells when total nucleated cells are expanded can include very few amplified cd34+5 progenitor cells. An average cord blood unit contains 2xl06 CD34+ progenitor cells (i.e., less than 1% of total nucleated cells) at a dose rate of lxl05 CD34+ cells/kg for a patient weighing up to 20 kg. A 5-6 fold expansion of a CD34+ progenitor cell therefore requires the clinical application of cord blood units in an average adult patient. Preferably, the expanded cells maintain pluripotency 10 (multipotency). A specific example of the present invention satisfies the currently known form of the deficiency 15 definition by providing a method for amplifying cord blood CD34+ progenitor cells in a bioreactor, as used herein, in vitro (ex_viv〇), meaning A method in which cells are removed from a living organism and propagated outside the organism. As used herein, with respect to the methods of the invention, cell expansion (ceU expansion) or 'expanding' It refers to a process of cell proliferation that is substantially devoid of fine 20 cell differentiation. The expanded cells thus maintain their cell renewal properties. "Progenitor cell" as used herein generally refers to an immature or undifferentiated cell that is self-renewing and can differentiate into a variety of different adult cell types. Hematopoietic progenitor 13 200916583 Certain types of cells may alternatively be referred to as stem cells. "Umbilical cord blood CD34+ progenitor cells" are cells expressing C34 antigen derived from hematopoietic stem cells in cord blood. They can cause cells of the bone marrow and/or lymphoid lineage. 5 mesenchymal stem cells are plurip〇tent blast cells found in bone marrow, corpus, dermis, and other locations. They can differentiate into a variety of tissue types. ), or "encapsulating" it means that the cell or cell is completely embedded within the support matrix by one. It is assumed that the size (e.g., the ratio of surface area to volume) enables nutrients, metabolites, cytokines, and the like to be easily diffused into/out of the beads to reach cells embedded in the beads... or cells The appearance of the beads is not particularly relevant. "Music culture container" as used herein means any type of container used to culture cells under non-static conditions. A dynamic culture vessel is typically maintained by a mixing or mixing mechanism. A homogeneous environment for cell growth, and variables such as temperature and pH can be monitored and controlled. A dynamic culture vessel typically contains one or more effective methods for maintaining stable environmental conditions within the container. (for example, it does not rely on diffusion for transporting nutrients in the container) and is capable of allowing continuous feeding of cultured cells. As used herein, "bioreactor" means a biological and/or biochemical process that is monitored and Devices developed under controlled environmental and operational conditions such as pH, temperature, pressure, nutrient supply, and waste removal. 200916583 Hematopoietic stem cells (HSCs) are stem cells and early precursor cells that cause all blood cell types. Including bone stalk (mononuclear ball and giant scorpion cells, sputum neutral ball, alkaline ball, sputum acidity Balls, red blood cells, megakaryocytes/platelets and certain dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells, certain dendritic cells). As stem cells, they are exposed to their ability to form multiple cell types. And their ability to self-renew. Individual HSCs have the ability to cause any type of end-stage blood cells. During differentiation, children and cells derived from HSC receive a series of commitment decisions' for 15 20 certain lineages In this way, the differentiation potential is maintained but the other is lost. The intermediate cells gradually become affected by their lineage potential until the final lineage-directed cells are produced. In humans, white blood cells (hematopoietic Nucleated cells contain a mixture of inflammatory cells. More than 99% of mononuclear cells are lineage-directed cells of differentiated cells. Hematopoietic stems and subtypes of progenitor cells (subtyPe and their lineage potential can be The various differences exist in their table and are defined by the fact that the CD3 4 is the cell surface ^^ Cell adhesion, and it is typically used as a non-differentiated hematopoietic stem and progenitor cells of birch 5-iff CD133 is another glycoprotein that is found in hematopoietic stem cells. Marker of the cell. CD38 is a glycoprotein found on the surface of the nucleus nucleus cells, which can be used as: a marker for '#differentiated cells. Non-lineage-directed and/or undifferentiated sputum and/ Progenitor cells typically contain low levels of markers (such as thy-1 lineage 15 200916583 CD71), which designate a more mature source ancestor population. A variety of different hematopoietic cell types can be identified by other markers, such as for bone marrow CD13 & CD33, for erythrocyte CD71, for B cell CD19, for megakaryocyte CD61, for mononuclear cell Mac-1 5 (CDllb/CD18) For granules Gr-1 or for T-cells CD3, CD4, CD5 and CD8. According to the invention, the CD34+ progenitor cells are preferably non-lineage-directed progenitor cells, such as CD34+ lin-, CD34+CD38- or CD34+CD133+ comprising less than 1% of total mononuclear cells. However, in an alternative embodiment of 10, the invention may also be used to amplify lineage-directed progenitor cells, such as CD34+CD33+ (bone marrow-directed progenitor cells), CD34+CD3+ (lymphoid-directed progenitor cells). And CD34+CD61 + (megakaryocyte-directed cells). The CD34+ progenitor cells used in the methods of the invention are derived from cord blood cells. In some embodiments, the cord blood sample can be subjected to a selection, purification or some sorting step to provide a sample enriched in CD34+ progenitor cells. A cell sample enriched with CD34+ progenitor cells is then used in the method of the invention, e.g., as a ligated blood cell used in the coating step. 20 Methods for purifying or extracting hematopoietic stem and/or progenitor cells from cord blood are known in the art. For example, a hematopoietic mononuclear ball is obtained from a blood sample by applying a blood sample to a Ficoll-Hypaque layer and an interfacial layer present between Ficoll-Hypaque and blood serum after density buffer centrifugation. The interface layer consists essentially of white blood cells present in the blood sample on 16 200916583. "Buffy coat cells" are used herein to mean the majority of the white blood cell population that can be found in the skin layer of the skin of a centrifuged blood sample, between the clear and red layers. For the avoidance of doubt, the blood-stained hemosphere cells that can be used in the method of the present invention can be obtained from any processing procedure. In other words, "skin hemocyte cells" means the type of cells found in the skin color portion of a centrifuged blood sample, and does not need to be the cells actually obtained by this method. Hematopoietic mononuclear cells, such as skin color hematopoietic cells (derived from umbilical cord 10 blood samples), can be used as umbilical cord blood cells in step (a) of a preferred embodiment of the method of the invention because they contain CD34+ Source progenitor cells. Alternatively, CD34+ progenitor cells can be further enriched, for example, by differential density centrifugation and/or immunoseparation, for example using immunomagnetic separation or flow cytometry/fluorescence-activated Cell-Classification (FACS). Antibodies that selectively target hematopoietic stem and progenitor cell types, such as monoclonal anti-CD34 and/or anti-CD38 antibodies, can be used in the selection/enrichment step. Such antibodies are commercially available and/or can be obtained by standard techniques. The medium used for cell growth to increase the number of fine cells in the support matrix structure (ie, amplification, wherein the cells undergo self-renewal by cell division) may be a medium that supports cell growth, ideally at a minimum or In the absence of cell differentiation. Various suitable maintenance media for hematopoietic stem cell expansion are known in the art, such as Stemline II (Sigma, St Louis, MO). The coating is applied to a support matrix of umbilical cord CD34+ progenitor cells according to the method of the invention of the present invention, in the method of the invention of the present invention, and a combination of a dynamic culture vessel can suppress the cell differentiation of the various media to be used together. For example, it is not necessary to include additional specific reagents (such as cytokines) in the medium that inhibits differentiation. Suitable environmental conditions for culturing progenitor cells to allow cell expansion are known in the art, for example, this step can be carried out at 37 ° C and 5% CO 2 . In order to allow amplification of CD34+ cells, the pH in the culture is preferably at least 6.7, more preferably 6.8 to 7.4. The omolarity is preferably from 0.25 to 0.35 mOsmol/kg, more preferably from 0.28 10 to 0.32 mOsmol/kg. In some embodiments, cytokines can also be included in the culture medium. In one embodiment, one or more cytokines that inhibit or protect against cell cell death (committed cell death) are used. In one example, the cytokine can be an early cytokine. In a further embodiment, the combination of cytokine in the medium and/or concentration may be different from the combination of cytokines in the beads (e.g., in a hydrogel). In one example, early acting cytokines such as, but not limited to, dry 2 cytokine (SCF), FLT3 ligand, interleukin-6, and interleukin-3 can be used. Thus, in a particular embodiment, the medium may contain one or more of the following concentrations of cytokines: 50 to 150 ng/ml of SCF, 50 to 150 ng/ml of Flt3, and 10 to 100 ng/ml of IL-3. And IL-6 at 10 to 100 ng/ml. More cytokines may be present in addition to or in lieu of one or more of the above cytokines in 18 200916583. For example, the cytokine may comprise one or more stem cell factors, thrombopoietin, and granule globule-community stimulating factors, each of which has a range of 1 to 1 ng/ml, such as 1 to 500 ng/ml, 10 5 to 100 ng/m b 1 to 50 ng/ml or 1 to 10 ng/mi. Although the use of cytokines is preferred in certain specific cases, the use of a face-to-face is less preferred. A high concentration of growth factor increases the cost of implementing the method. Also, in some specific examples, an excessive concentration may potentially mislead cells for expansion and differentiation into abnormal pathways. In a particular embodiment of the invention, it is advantageous that a lower concentration of cytokines (as compared to those employed in the prior art methods) can be used. For example, in a particular embodiment, the concentration of each cytokine can be 200 ng/ml or lower, 1 ng/ml or lower, 5 ng/mi or lower, 20 ng/ml or more. Low, or 1〇ng/ml or lower, such as} to 15 _ ng/m b 1 to 5 ng/ml or 1 to 10 ng/ml. Static culture vessels, such as a tissue culture pan, allow the cells to grow only 2. and may suffer from such requirements as lack of mixing, poor control options, and related customary feeds. The step of using a bioreactor, which provides a dynamic culture system, along with controlled culture conditions, allows the fines to be expanded - closer to those in a 3-dimensional environment of living organisms. In certain specific aspects of the invention, the cells are grown and cultured in a flask or a rotating flask bioreactor. The rotating bioreactor provides a homogeneous environment and is easy to produce, allows sampling, and monitors control money. Typical modes of operation include batch, feed_200916583 batch, and perfusion mode (medium exchanged with cell retention by an external filtration module or internal device (such as a filter). The spinner flask can be a plastic or glass bottle with a central magnetic stir bar and side arms for the addition and removal of cells and culture medium, and enriched with C〇2i air for 5 to inflate. The inoculated spinner flask was placed on a stirrer and incubated under appropriate culture conditions. For example, the culture can be stirred at a speed of 1 Torr. 25 Torr per minute, preferably 30-100 rpm, and optimally at 50 rpm. A rotator designed to control a culture volume of 1-12 liters and a rotating flask system is commercially available [such as Coming ProCulture System (Coming, Inc.,

Acton, MN). The rotating flasks are typically equipped with probes for monitoring pH, temperature, oxygen and C〇2 saturation in the culture medium, levels of metabolites (such as glucose, nitrogen, amino acids, etc.), as well as in fluids. Fluid communication, optionally assisted by a screw pump (with fresh supply of 15 bases, gas, specific nutrients and analogs, and waste removal) so that the medium can be removed or supplemented to The optimal conditions for stem cell expansion are maintained at a predetermined rate. Shear stress and turbulent eddies are sometimes a consideration for stirred flask bioreactors. Suitable suspension culture conditions for carrying out the cell culture method of the present invention can be achieved using a low shear stress, high mixing, dynamic environment. This allows sufficient nutrients and gases to pass through the support matrix structure used. The dynamic laminar flow produced by a rotating fluid environment is an efficient method for reducing nutrient and waste diffusion limitations while minimizing shear stress. Rotating wall containers have been used for in vitro cell growth of a variety of different cell types (see, for example, Vunjak-Novalovic et al, J Orthop Res 1999; 17: 130-38, Rhee, et al, In Vitro Cell Dev 2001; 37: 127-40, Licato et al In Vitro Cell Dev,

5 2001; 37: 121-26 and pei, et ai, FAseB J 2002; 16: 1691-94). Thus, in accordance with a preferred embodiment of the invention, the bioreactor is a rotating wall vessel bioreactor. Suitable rotating wall vessel bioreactors are well known in the art, such as HARV (e.g., NASA HARV), RWV bioreactor, Synthecon's Roller ίο Cell, and RCCS-1 (Synthecon Inc, Houston TX), European Space Agency bioreactor (Fokker, Netherlands), or other systems that simulate microgravity or perfusion, such as airlift bioreactors, and various types of spinners from Coming (Coming, Inc., Acton, MN). In a preferred embodiment, the RWV is a 15 HARV (Synthecon Inc, Houston TX) from Synthecon. In another embodiment, the bioreactor is a NovaPodTM bioreactor from NovaThera Limited, Cambridge UK, for example, as described at http://www.novathera.com, such as http://www.novathera.com http://www.novathera.com http://www.novathera.com www.novathera.com/documents/NovaPod I Brochu 2〇re Medica 07.pdf. Although the agitated bioreactor may produce shear stress, NovaPod has reduced shear stress. Shear stress can also be reduced by coating, which provides additional protection to the cells without reducing mass transport. The support matrix used for coating is permeable to allow diffusion and mass transport of nutrients, metabolites, and growth factors. Coated 21 200916583 Cells (a ceU) or cells within a support matrix can be provided in the form of a bead, such as a generally spherical bead. One or more progenitor cells can be coated in various support matrix structures, such as a bead. Typically, each bead may contain from 100 to i million living cells, from 53,000 to 300,000 cells, from 3000 to 2 cells, from one cell, from 5 to 200,000 cells, from 5000 to 50 inches. One cell, 10,000 to 300,000 cells, 20,000 to 200 cells, each cell, approximately 5,000 or approximately 200,000 cells per bead. In 2D conventional cultures, the cell size is 15 μιη ίο (0.0015 cm) and the well plate size is 1.2 cm2. The actual cell density can be 10,000 cells/0.0018 cm3. In contrast, assume that 5000 cells per bead are used. 'Assume that the diameter of the beads is 2.5 mm, and the actual cell density in the 3D culture is 5000 cells/〇〇36 cm3. Compared to 2D, there is about a 40-fold lower fine cell density in this 3D culture. In some embodiments of the invention, the use of each bead (2.5 mm diameter) 5000/cell allows for proper cell expansion. Higher densities (such as parent beads 20,000 or 2 〇〇, 〇〇〇 cells) can be used to increase amplification rates by a factor of 15 to 30, respectively. Alternatively, in some embodiments, amplification at a lower density of 20 may be preferred from the standpoint of the low number of available cells derived from the umbilical cord sample. It is particularly preferred that the support matrix structure, such as beads, be formed from a suitable support matrix material that remains intact during the incubation period. Cells or cells (ceUs) encapsulated within a support matrix can be placed in a culture vessel, such as an RWV bioreactor 22 200916583 (Synthecon, US A) or other simulated microgravity or perfusion bioreactor And it is cultivated in the maintenance and/or differentiation medium without significant damage for a prolonged period of time. Preferably the support matrix material consists of a gel forming polysaccharide or 5 comprises a gel forming polysaccharide such as agarose or alginic acid (typically ranging from about 0.5 to about 2% w/v, preferably from about 0.8 to about 1.5% w/v, more preferably about 0.9 to 1.2% w/v). The matrix may be composed of alginic acid alone or may further comprise a component such as gelatin (typically from about 0.05 to about 2% v/v, such as from 0.05 to about 1% v/v, from about 0.08 to about 0.5% v/v). , or about 1% v/v). Containing gelatin helps to create a uniform bead size and helps maintain structural consistency. The inclusion of gelatin in the alginic acid-supporting matrix beads enables shrinkage of the cell medium and packing of the scaffolding material. In a preferred embodiment, the support matrix material comprises a) alginate 15 and mercaptocellulose, or b) alginic acid to fibronectin, or c) alginic acid, methylcellulose, and fibronectin. The amount of each of these ingredients can be varied to achieve the consistency and hardness of a desired support matrix, which is most beneficial for cell expansion. Preferably, the support matrix comprises 0.01 to 1% v/v mercapto cellulose, more preferably 0.1 to 1.0% v/v methyl fiber 20, and most preferably about 0.1% v/v mercapto cellulose. . Preferably the support matrix comprises from 0.5 to 500 gg/ml fibronectin, preferably from 1 to 100 pg/ml fibronectin, more preferably from 1 to 50 pg/ml fibronectin and optimally about 50 pg /ml fibronectin. In a particularly preferred embodiment, the support matrix comprises 0.05 to 0.5% v/v 23 200916583 methylcellulose and 1 to 100 Mg/ml fibronectin. Alginic acid is a water-soluble linear polysaccharide extracted from brown algae and consists of parenting blocks with 1-4 linkage # CX-L-glucose via acid and p_D_mannose acid residues. Alginic acid forms a gel with most of the 1-5 and polyvalent cations, although Ca2+ is the most widely used. λ field. The support matrix is in the form of beads containing a single cell, which may be, for example, from about 2 to 15 micrometers in diameter, preferably from about 4 to about 1 inch. suddenly. The beads containing the complex & 1〇 cells may have an average diameter such as 0,1 to l〇mm, 〇.5 to 10 mm, 1 to i〇mm,! To 5 mm, from about 2 Torr to about 25 mm' to about 2.3 mm or about 2.5 mm. In certain aspects of the invention, the preferred support matrix can be readily dissolved to release cells without the use of trypsin 15 (trypSmiSation). In the case where the support matrix is removed to release the cells, hydrogel matrices such as alginic acid and alginic acid-based matrices are preferred because they can use sodium citrate and sodium carbonate solution. Easy to be dissolved. In addition to the selective freezing step after the coating step (a), a method or use according to the invention may further comprise frozen coated cells for storage, for example in cell culture and amplification step (c) after that. The coated cells can be frozen using standard procedures and can be frozen in the maintenance or differentiation medium in which they are cultured. A suitable method for cold/east coated cells involves the use of a slow cold beam manipulation step 24 200916583 (as described by Stensvaag et al (2004) Cell Transplantation 13 (1): 35) cryopreservation in disulfoxide (DMS〇). The method of the invention may further comprise releasing a cells or cells from the support matrix. The present invention thus provides 5 cells (a cel1) or cells thus obtained. If alginic acid or an alginic acid-based matrix is used for coating, the release of cells can be achieved by dissolution of alginic acid. These mild solubilization methods may affect the behavior of cells in long-term culture favorably r compared to standard enzyme methods (such as trypsin action.) In some embodiments, the methods of the invention may involve further induction. The step of differentiation of the expanded cell population. Differentiation can be induced while the cells are still being coated, or alternatively after releasing the cells from the support matrix. In some embodiments, the removal of the coating facilitates Differentiation of cells. Certain cell lines undergo spontaneous 15 differentiation after the cell division cycle in which growth is maintained, particularly in the case where differentiation is not inhibited. Any medium for differentiation is suitable for use in the method of the present invention. The medium % may be similar to the one used in the amplification step, except that the substance to be differentiated is included in the specific example 10 of the culture beauty used to culture/amplify the source progenitor cells, and the substance is not included in Differentiation medium. Suitable: 20 cell differentiation conditions may include differentiation of CD34+ progenitor cells: stimulation. The stimulation of differentiation may be related to differentiation. For example, stimulation of the lymphoid hematopoietic lineage. In addition to the differentiation step, the conditions can be similar to those of the user in the amplification step. Brother, for example, therefore the differentiation and amplification steps can be in the same container 200916583 = object The method of integration of the differentiation method is suitably carried out in a burned suspension, and is carried out in a coated suspension culture. In the amplification phase, the cell population is split in #6, the 徂 cell divides and the number of cells is increased, so that the fine-formed one-eight matrix is formed. Formed within the structure. The coated cells are then differentiated into the inner part of the structure: the second two: there are 3d matrix cells can be followed by the dimension = go, the differentiation or terminal differentiation of the empowerment of the heart cell community in support == shape : making the number of cells 10 15 20 2 In a specific example, a reagent for inducing differentiation can be added to a cell population in a container, including but not limited to Ca2+, EGF, Ava-FGF, beta _FGF, pDGF , keratinocyte growth factor (KGF), TGF beta, cytokines (eg 1L_1 Ava, IL-1 beta, IFN-gamma, TFN), retnolc acid, transport Ferritin , hormones (eg, androgens, eosin, insulin, prolactin, triiodothyronine, hydrocorticosterone, dexamethasone), sodium butyrate, TPA, DMS0, NMF, DMF, matrix elements (eg collagen, mucin) The heparin sulfate, or a combination thereof. The duration of the amplification and differentiation steps is not particularly limited. For example, the amplification step can be maintained for at least 1, 5, 10, 30 or 100 days, for example 1 to 30 days. The cultured and expanded cell population prepared according to the method of the present invention or the differentiated cells obtained therefrom can be used as a medicament, for example, in a regenerative therapy. Typically, such cells 26 200916583 are administered to a patient after removal of the support matrix (i.e., after release of the cells from the coating). In general, regenerative therapies include a wide variety of treatment steps in which a tissue or organ of the body is enlarged, repaired, or replaced with a 5 transplant (enSraftment) of a desired cell population (such as a stem cell or a source progenitor population). , transplantation or perfusion. In a preferred embodiment of the invention, the cells are used to treat fluid disorder A or repair bone marrow after high dose chemotherapy. The expanded population of cells according to the present invention can be used as an autologous or homologous, including paired or mismatched HLA-type hematopoietic transplants. 1〇 Cultured cells can be used to repair tissue and organ damage caused by disease. In this embodiment, a patient can be administered a cultured progenitor cell to regenerate or repair a tissue or organ that has been damaged as a result of the disease, for example, to enhance the immune system after chemotherapy or radiation, or in the myocardium Repair the heart tissue after the infarction. Cultured 15 cells can be used to amplify or replace bone marrow cells in bone marrow transplantation. <Human autologous or allogeneic bone marrow transplantation is currently used as a therapy for diseases such as white disease, lymphoma or other life-threatening diseases. However, the disadvantage of these procedures is that a large number of donor bone marrow must be removed to ensure that there are enough cells for transplantation. The expanded cell 2〇 population according to the present invention can provide stem cells and progenitor cells which are reduced in response to the big time. Thus in one embodiment, the expanded population of cells can be used in a complementary treatment other than chemotherapy. Most chemotherapeutic agents used to dry and destroy cancer cells act by killing all proliferating cells (ie, cells that enter cell division on 27 10 20 200916583). Because of bone, and one of the hyperplastic tissues, hematopoietic stem cells are most likely to be injured or destroyed and as a result, blood cell production is reduced or terminated. The ground is terminated to allow the patient's hematopoietic system to re-open the blood of the therapy. It may take time for the cells to be stopped, or a few to accumulate and increase the number of white blood cells to the acceptable level of chemotherapy. (# Again, the bone (four) cells are destroyed by the blood cells between the chemotherapy treatments. Will regenerate, however, cancer has $ between growth and may become more chemotherapeutic because of natural selection. Therefore, longer chemotherapy is given and the duration between treatments is shorter, the greater the chance of successfully killing cancer In order to shorten the time between chemotherapy treatments, the expanded source cell population generated according to the present invention can be introduced into a patient. This treatment can reduce the time during which the patient will exhibit a low blood count, and will therefore Allowing early recovery of chemotherapy therapy. The invention also encompasses pharmaceutical compositions comprising an expanded population of CD34+ progenitor cells produced according to the method. These cells can be mixed with, or as a mixture with, other stem cells Use for transplantation or other uses. The expanded population of cells (typically after release from the support matrix) can be rendered unfrozenly , or frozen for later use. If the cell population is to be frozen, a standard cryopreservation (eg DMSO, glycerol, Epilife (registered trademark) Cell Freezing Medium Cascade Biologies) is added to it before it is frozen The enriched 28 5 10 15 20 200916583 cell population. In some embodiments, - or a plurality of source cell populations can be delivered to a patient in need thereof. In a specific embodiment, the invention provides a method of treating or preventing - an individual - a disease or disorder, comprising the need for treatment or prevention - (iv) (d) - therapeutically effective amount (four) progenitor cells (obtained from the methods defined herein) ). In another embodiment, the invention provides a method of treating or preventing a disease or disorder in a subject, comprising the need for an individual to administer or treat a therapeutically effective amount of CD34+ obtained by the method of the invention. Source progenitor cells. In yet another embodiment, the expanded population of cells can be used to treat any disease, condition, or condition that is caused by inflammation or associated with inflammation. Inflammation can occur in any organ or tissue, such as muscle; the nervous system, including the brain, spinal cord, and peripheral nervous system; vascular tissue, including heart tissue, · pancreas; organs of the intestine or other digestive tract; lung; kidney; liver, genitals m tissue (endothelial tis (four), or endodermal tissue. Cell populations can also be used to treat immune-related disorders, particularly autoimmune disorders, including those associated with inflammation. Therefore, in some specific cases The present invention provides a method for treating an individual having an autoimmune disease or condition, comprising administering to the individual an effective number of progenitor cells derived from the method, wherein the disease mouth or disorder " But not limited to diabetes, amylotrophic lateral sclerosis (ALS), myasthenia gravis 29 200916583 (myasthenia gravis), diabetic neuropathy or lupus (diabetic neuropathy or lupus), acute or chronic allergies (eg Jilang Sexual allergies, food allergies, allergies to specific antigens. Others can be treated Diseases include aplastic anemia, myelodysplasia, myocardial infarction, seizure disorder, multiple sclerosis, stroke, low jk pressure, cardiac arrest (cardiac arrest) ), ischemia, inflammation, age-related loss of cognitive function, radiation damage, cerebral palsy, neurodegenerative disease, Az Hyperthermia, Parkinson's disease, Rie's disease, AIDS dementia, amnesia, amyotrophic lateral sclerosis (ALS), ischemic nephropathy, brain or spinal cord trauma, heart-lung bypass, glaucoma, retinal defect Retinal ischemia, retinal trauma, lysosomal storage 15 disease, such as Tay-Sachs, Niemann-Pick, Fabry ( Fabry's), Gaucher's, Hunter's, and Hurler's syndrome, as well as other gangliosidoses, Mucopolysaccharidoses, glycogenoses, congenital 20 metabolic abnormalities, adrenoleukodystrophy, cystic fibrosis, glycogen storage disease, thyroid hypoenergy Hypothyroidism, sickle cell anemia, Pearson syndrome, Pompe's 30 200916583 disease, phenylketonuria (PKU), porphyrias ), maple syrup urine disease, homocystinuria, mucoplysaccharidenosis, chronic granulomatous disease and tyrosinemia , Taiyi Sa's disease, cancer, tumor or other pathology or new condition. In other embodiments, the cells can be used to treat any injury caused by trauma, particularly inflamed wounds. Examples of such wound-related conditions include central nervous system (CNS) injury, including damage to the brain, spinal cord, or tissues surrounding the CNS injury to the peripheral nervous system (PNS); or damage to any part of the body. This trauma can be caused by an accident or can be a normal or abnormal result of a medical procedure such as surgery or an artery such as a tube • surgery. The wound can be the result of a fracture, destruction or blockage of a blood vessel, such as a stroke or phlebitis. In a particular embodiment, the cells can be used in autologous or allogeneic tissue regeneration or replacement therapy or procedures including, but not limited to, corneal epithelial defects, cartilage repair, facial dermabrasion, Mucosal membranes, tympanic membranes, intestinal linings, neurological structures (eg retina, auditory nerve in the basement membrane, olfactory nerve of the olfactory epithelium), burns and targets Treatment of wound traumatic wounds or wound healing associated with reconstruction of other damaged or rickets of organs or tissues. The dose of cells administered to a subject in accordance with the present invention will depend on the size of the individual 3 1 200916583 and the nature of the condition in need of treatment. Typically, a patient who is perfused with a stem cell (e. g., with respect to a bone graft) receives one unit of cells, one of which is about ίο9 nucleated cells (corresponding to 1-2 X 108 stem cells). According to the invention, a patient is preferably treated with at least 5 105 CD34+ progenitor cells/kg, for example a total of 106 to 107 CD34+ cells, or about 7 x 106 CD34+ progenitor cells for a typical male adult. . The progenitor cells can be administered to a patient in any pharmaceutically or medically acceptable manner, including by injection or infusion. Such cells 10 or supplemented cell populations may contain, or be included in, any pharmaceutically acceptable carrier. The cord blood or cord blood-derived stem cells can be carried, stored, or transported in any pharmaceutically or medically acceptable container, such as a blood bag, shipping bag, plastic tube or bottle. [Embodiment] Example 1 Cell source: Hematopoietic cells are CD34+ progenitor cells (HPC) derived from sputum blood (UCB) contained in the blood layer of the skin color. 20 Collection and treatment: Human umbilical cord blood is obtained from cord blood after normal term delivery (by informed consent). Cord blood is collected in a standard 250 mL blood bag (containing CPD anticoagulant) and used within 24 hours postpartum 32 200916583

Sep ax (Bio safe) technology is processed for use in UCB. The early blood (blood layer of the skin color) was cooled in 1% DMSO using a rate-controlled cold bundle and stored in the liquid phase of liquid nitrogen. The cells were quickly unrolled (using a 37 ° C water bath) to 5 Stemline II serum-free medium (Sigma, St. Louis, MO, USA) and washed 3 times in Stemline II medium before using them. To remove DMSO. Cell counting, flow cytometry, and colony formation analysis were performed at this point. 10 Alginic acid/fibronectin/methylcellulose coating Alginic acid was mixed with 0.1% v/v thioglycol and 50 pg/ml fibronectin. One is for each chamber 1 〇 7 living cells are coated in a whole - part of 500 beads 'for example, each bead 2.OxlO4 living cells. Cover the medium and supplement the Stemline II with Stemspan CC100 cytokine mix. * Chlorophyll, streptomycin and jitamycin are used in the culture medium to prevent the growth of bacterial contamination in cord blood units. Umbilical V blood-stained hematopoietic cells were counted and X 1.6 X 1 〇 cells/mL were resuspended in sterile sterile low-viscosic alginic acid at room temperature (21) (this specific product is a kind of money, hydrophilic, Colloid and glycan acid, mainly consisting of a mixture of anhydrous mannose mannose road and Q1% (v/v) pig gelatin (called 脱, υ κ) (all in this solution in ?38, yang 7.4) solution Use a screw chest (]^〇(^11}_1, 33 200916583

Amersham Biosciences, UK), a flow rate producing single droplets, a drop height of 30 mm (sterilized by tubing and then sterilized with 1 M NaOH for 30 minutes and washed with sterile PBS) 3 times) 'Cell gel solution 5 is passed through a peristaltic pump and uses a 25 gauge needle (Becton)

Dickinson, UK) Instilled into sterile, room temperature (21. 〇, 100 mM calcium carbonate solution (CaCl2; Sigma, UK) and 1 mM N-(2-hydroxyethyl) in distilled water (pH 7.4) - Piper-N-(2-ethanesulfonic acid) (HEPES; Sigma, UK). The cell-gel solution condenses immediately after contact with the CaCl2 solution, forming a spherical bead (2.3 mm diameter after bulging) The beads were maintained in a gently stirred CaCl 2 solution at room temperature (21 ° C) for 6-10 minutes. The beads were washed 3 times in PBS and placed in maintenance medium for each alga The optimum total cell count for acid beads can be in the range of 20, 15 cells. However, it is important to confirm that each bead contains the optimal number of CD34+ progenitor cells rather than the total cell count. Cord blood color blood cell i layer The cells were therefore evaluated for the percentage of total CD34+ cells in an attempt to normalize the number of CD34+ progenitor cells per bead. 2 Alginic acid beads dissolved

A sterile depolymerization buffer was used to dissolve the beads consisting of 5 mM mM dihydrate (Fluka, UK), 77 mM sodium bromide (UK) and 10 mM HEPES. Lysis buffer (Ca2+-depletion) was added to the pbs washed beads 34 200916583 while gently stirring for 15-20 minutes. The solution was centrifuged at 400 g for 10 minutes and the pellet was washed with PBS and centrifuged again at 300 g for 3 minutes. 5 cytokine combination of these cytokines when used at a working concentration of 100 ng / mL recombinant human Flt-3 ligand; 100 ng / mL recombinant human stem cell factor; 20 ng / mL recombinant human IL-3; 20 ng/mL recombinant human IL-6. 10 Planting Bioreactor The coated cells were cultured in 50 mL of Stemline II medium (supplemented with Stemspan CC100 cytokine cocktail and antibiotics) in a HARV bioreactor. The speed of the bioreactor was 17.5 RPM, and the cells were cultured at 37 ° C in 5% CO 2 in air. Feeding the coated cells to the bioreactor Day 0 is the time when the bioreactor is started. The bioreactor was fed on days 20, 5 and 7 and harvested on day 10. The old medium was withdrawn and 50 mL of fresh, warm Stemline II medium (supplemented with Stemspan II cytokine mixture) was forcefully injected into the beads. The beads are resuspended and reintroduced into the bioreactor. 35 200916583 Harvesting bioreactor The bioreactor was harvested on day ίο. The beads and medium are removed from the bioreactor chamber into a centrifuge tube and the cells are released by dissolving the alginate/methylcellulose/fibronectin. The cells were centrifuged at 5 1500 rpm for 5 minutes, the old medium was poured out and replaced with fresh medium. Cells were evaluated using flow cytometry and colony forming unit granulocyte macrophage (CFU-GM) analysis. This colony forming unit particle globular macrophage (CFU-GM) assay is often used to assess the appropriateness of the number of ancestral ancestors on the cell population for bone marrow transplantation. Analytical Technique Type Assessment · In order to depict the resulting culture population, the aliquots of cells are placed on a slide (cytocentrifuge, 15 Shandon, Runcorn, UK), fixed and in Mege It was dyed in the May-Grunwald and Giemsa stain. CFU-GM analysis and CD34+/lin- (lineage negative) flow cell measurements were performed using standard operating procedures according to the instructions of the manufacturer. CD34+ progenitor cells were identified using a labeled anti-CD34 monoclonal antibody 20 (commercially available). In vitro expansion of total nucleated cells (TNC), CD34+ din-cells, and CFU were reported as cumulative numbers; the number of cells per ml 36 200916583 was multiplied by the final culture volume, or as a multiple-expansion Increase; the cumulative number divided by the number of starting implanted cells. The CFU frequency is calculated as the number of colonies divided by the number of cells. 5 Statistics The following statistical tests were used: Wilcoxon Rank Test (Wilcoxon Rank Test) was applied to the differential supply parameters between the experimental study populations. All of the tests applied were two-tailed, and 5% or less of p値 was considered to be statistically significant. The data was analyzed using the SAS ίο software (SAS Institute). Example 2 In yet another embodiment, the method was performed as in Example 1, except that in the flow cytometry step, CD34+/CD133 + 15 was used as a combination of markers. Primary antibody-CD133 monoclonal antibody can be used to identify CD133+ cells. Examples 3 and 4 In a further embodiment, the method is carried out as described in a) Example 1 in 20 and b) Example 2, except that the coating medium is PBS instead of Stemline II and no cytokines are Addition to the medium Example 5 to 8 37 200916583 In a further embodiment, the method is carried out as described in each of Examples 1 to 4 except that approximately 200,000 viable cells per bead are coated . 5 Example 9 In this example, hematopoietic CD34+ progenitor cells were expanded using a method similar to that described in Example 1 with the following modifications. Human umbilical cord blood (hCB) mononuclear cells were harvested from hCB units by density gradient Ficoll 10 centrifugation. CD34+ progenitor cells were isolated using a magnetic cell sorter (MACS) and only hCB CD34+ cells with a purity greater than 95% were used. Alternatively, CD34+'s source ancestors can be obtained coldly from a commercial source. The isolated hCB CD34+ source ancestor was coated in a hydrogel (containing 15 (w/v) ° / alginic acid and 1 (v / v)% gelatin in PBS), in a serum-free In the expansion medium. The cells were coated at a cell density of 5,000 cells per hydrogel bead, each having a diameter of about 2.5 mn. The coated cells were allowed to expand in a NovaPodTM bioreactor system for 10 days. 2〇 The CD34+ source progenitor coated in the hydrogel beads was cultured at a cell density of 5000 cells/hydrogel for 10 days and was fed on days 0, 4 and 7. Cytokine stem cell factor (SCF), thrombopoietin (TP0), and granule globule-community stimulating factor (GCSF) were added to the medium (StemLine II) at a concentration of 1〇ng/mi on day 3 and at 38 200916583 Days 4 and 7 were performed under these conditions with two individual experiments at 100 ng/m (A1 and A2 in Table 1) and the results were averaged. A 6.7-fold increase in total cell number with high cell viability (90%) was achieved (see Figures 2 and 3). The coated CD34+ source progenitor cultured in the same culture but lacking cytokines (Experiment B in Table 1) showed a lower degree of total cell expansion. After 10 days of expansion, the percentage of CD34+ source progenitors decreased from 96% to 76%, as confirmed by flow cytometry (Figure 1), suggesting a net 5.3-fold increase (Table 1). On day 10, the cells in the beads were cultured for another period without medium renewal and allowed spontaneous differentiation, indicating that they maintained proliferative potential (Fig. 4). Therefore, the hBC CD 34+ source ancestors were 10 days of 3D coating and NovaPod bioreactor culture, and were coated in alginic acid and gelatin in serum-free StemLine II medium with low concentration (l〇〇ng/mL). The 15 surviving cytokines (TPO, G-CSF and SCF) caused a 6.7-fold increase in total nucleated cells with a 5.3-fold increase in CD 34+ source ancestors. 39 200916583 Table 1 Experimental medium Total nucleated cell expansion CD34+ cell expansion (approximately 75.72% purity) A1 with 10 mg/mL on day 0 and 100 ng/mL on day 4 and 7 with alginic acid/cell StemLine II of the mixture SCF, TPO & G-CSF Day 4 = 4600 ± 875 Day 7 = 8145 ± 1055 Day 10 = 36015 ± 4063 1 day amplification = 7.2 times (Day 0 = 5000, 10th) Day = 36015) 10 days amplification = 5.9 times (Day 0 = 4786.5, Day 10 = 27990.858) A2 with 10 mg/mL on day 0 and 1 OOng/mL on day 4 and 7 with alginic acid / Cell Mixture SCF, TPO & G-CSF StemLine II Day 4 = 4500 ± 912 Day 7 = 8000 ± 714 Day 10 = 31278 ± 1734 10 Days Amplification = 6.3 times (Day 0 = 5000, 10th) Day = 31278) 10 days amplification = 5.0 times (Day 0 = 4786.5, Day 10 = 23683.7016) A (l & 2) averaged 10 mg/mL on day 0 and 1 OOng/mL on days 4 and 7. StemLine II with alginic acid/cell mixture SCF, TPO & G-CSF Day 4 = 4550 ± 830 Day 7 = 8073 ± 838 Day 10 = 33646 ± 3844 10 Days Amplification = 6.7 times (0 Day = 5000, day 10 = 31278) day amplification = 5.3 times ( Day 0 = 4786.5, Day 10 = 25476.7512) B Only StemLinell Day 4 = 2450 ± 957 Day 7 = 3800 ± 849 Day 10 = 5700 ± 572 10 Days Amplification = U4 times (Day 0 = 5000, Day 10 2, 5700) 10-day amplification = 1.lx reduction (Day 0 = 4786.5, Day 10 = 4316.04) 40 200916583 5 et al. (1994). Curr. et al. (1999). Exp et al (1999a). Int. et al. (1997). Exp. References Andrew, RG, (3): 187-196. Bachier, CR, 615-623. Chabannon, C·, 511-518. Gehling, UM , 1125-1139.

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Wolff, S. N. Bone Marrow Transplant 2002; 29 (7); 545-52. 10 All of the publications mentioned in the above specification are incorporated herein by reference. The various modifications and variations of the methods and products of the present invention are apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with a particular preferred embodiment, it should be understood that the claimed invention is not limited to the particular embodiment. Moreover, any of the various modifications of the modes described for the application of the present invention (which are apparent to those skilled in the art) are intended to fall within the scope of the following claims: 简单 [Simple Description] 20 All aspects of the invention will be described with reference to the following non-limiting specific examples and the drawings by way of example only, wherein: Figure 1 shows the flow cells of the CD34+ cell population prior to cell expansion. Results of the measurement; * Figure 2 shows a type image of cell growth within the beads after culturing the CD34+ cell population with cytokines 1 day 42 200916583. Figure 3 shows live/dead staining images of CD34+ cell populations at different times after the start of cell culture in the presence or absence of cytokines to indicate cell viability. 5 Figure 4 shows a type image of the 3D cell population after cell expansion without updating the cell culture medium. Differentiation was discovered to indicate that the potential has been maintained. [Main component symbol description] 10 None 43

Claims (1)

200916583 X. Patent Application Range: 1' A method for in vitro expansion of cord blood CD34+ progenitor cells, comprising: (&amp;) coating cord blood cells in a support matrix; (b) coating the fine 5 The cells are grown into a dynamic culture vessel; and (c) the cells in the dynamic culture vessel are cultured under conditions that permit CD34+ source sputum cell culture. 2. If you apply for a patent scope! The method of the item wherein the cells are derived from human umbilical cord blood. 10 15 3. 4. 5.6. If the patent application range is 1 or 2, it is maintained in a serum-free medium. Any of the foregoing patent applications is a bioreactor. The method of the item wherein the cells are cultured, wherein the dynamic culture is carried out as in the method of claim 4 of the patent scope of the rotating wall container bioreactor. The bioreactor is a square HARV bioreactor as in claim 5 of the patent application. The towel (four) reactor is - if any of the scope of the patent application contains - water_. (4) The heart of the support matrix package The method of any of the claims is directed to alginic acid. Wherein the support matrix package 9. Any one of the preceding claims includes a fibronectin/ricin/, wherein the support matrix comprises any one of the scope of the patent application containing methylcellulose. And the method wherein the supporting substrate lh is in accordance with any one of the preceding claims, wherein the supporting substrate is in the form of 44 5 15 20 200916583 ^ body particles.养丄ί:;Special: 任-item method, in which the cells are cultured (4) application;:;; c medium. A method of early action of cells = wherein the cytokines are 14. = y: = 13 terms, wherein the early effects are finely derived from a population consisting of stem cell factors and interleukin 3.素素 6 15 · As claimed in the scope of the uu 16: skin culture in - cell-free hormone culture Γ: wherein the two special = any one of the methods, wherein the cells in the presence of metal chelators Be selected. 17. The method of cold-blowing coated cells after the second step (8) of the aforementioned patent application further comprises the method of step 18. 第?, the step further comprises the step of sterilizing in the step (8) Covered cells, and unwrapped coated fine! 9. As mentioned in the foregoing patent scope, the item is collected 72 hours after the cord blood. The 4 cells are in the package. Any of the methods, cultured in the presence of mesenchymal stem cells. - The medium cell is in a process of any one of the preceding claims, wherein the substrate is released into the coated cell. An amplifying group obtained by the method of any of the aforementioned patent claims 45 5 10 20 200916583 An amplified population of gray CD34+ progenitor cells. 23. A dynamic culture vessel containing umbilical cord blood cells coated on a support matrix. A dynamic cell culture vessel of 23 patents, wherein the dynamic cell culture vessel is a rotating wall vessel bioreactor. 25. The dynamic culture vessel of claim 23, wherein the support matrix comprises a hydrogel. The dynamic culture container according to any one of claims 23 to 25, wherein the cord blood cells comprise CD34+ progenitor cells. 27. The expanded cell population of claim 22, for use in regenerative therapies. 28. Use of an expanded population of cells as claimed in claim 22 for the preparation of a medicament for regenerative therapy. &amp; 29. A method for treating a subject in need of a regenerative therapy, comprising: 2 administering the individual as a coated group of claim 21: a cell population of claim 27 to 29, Use or side: wherein the regenerative therapy comprises treatment - blood transfusion or chemistry - related bone marrow loss.咏 31.: The method 'includes (4) coating the cord blood cells with - support, and (b) cold rolling the coated cells. </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> The method of claim 1, wherein the cytokine package 46 200916583 comprises one or more of a stem cell factor, a thrombopoietin, and a particle ball-community stimulating factor. 34. 5 35. 36. If the patent application scope is 3000 to 300,000 cells per bead density is coated. The method of U or 34, wherein the ten sentences are only 1 to 1 mm. The method of claim 12, wherein each cell is activated to a concentration of 10 ng/ml. Susie 47