KR20130015481A - A method for in vitro large-scale production of red blood cells and storage thereof - Google Patents

Abstract

The present invention relates to an in vitro mass production method of red blood cells. More specifically, the present invention is to culture the erythrocyte progenitor cells in accordance with the conditions of the mineral (stroma-free, serum-free and plasma-free) medium and / or temperature change conditions according to differentiation time It relates to a method for producing large amounts of red blood cells in vitro.

Description

A method for in vitro large-scale production of red blood cells and storage

The present invention relates to an in vitro mass production method of red blood cells. More specifically, the present invention is to culture the erythrocyte progenitor cells in accordance with the conditions of the mineral (stroma-free, serum-free and plasma-free) medium and / or temperature change conditions according to differentiation time It relates to a method for producing large amounts of red blood cells in vitro.

In many countries, the supply of red blood cells for transfusions is scarce due to aging populations and increasing medical needs. And transfusion infection causes serious problems in the use of red blood cells. Such transfusion infections include viral and bacterial contamination such as human immunodeficiency virus, hepatitis B virus. Emerging pathogens such as blood-borne tropical diseases and variant Creutzfeldt-Jacob disease due to increased overseas travel are also problematic. These factors increase the need for artificially produced red blood cells (Greenwalt, TJ; Zehner Sostok, C .; Dumaswala, UJ Studies in red blood cell preservation. 1. Effect of the other formed elements.Vox Sang. 58: 85-89; 1990; Olsson, ML; Clausen, H. Modifying the red cell surface: towards an ABO-universal blood supply.Br. J. Haematol. 140: 3-12; 2008).

As a possible substitute for donated blood cells, blood preparation from various stem cells has been studied (Giarratana, MC; Kobari, L .; Lapillonne, H .; Chalmers, D .; Kiger, L .; Cynober, T. ; Marden, MC; Wajcman, H .; Douay, L. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.Nat.Biotechnol. 23: 69-74; 2005; Neildez-Nguyen, TM; Wajcman, H ;; Marden, MC; Bensidhoum, M .; Moncollin, V .; Giarratana, MC; Kobari, L .; Thierry, D .; Douay, L. Human erythroid cells produced ex vivo at large scale differentiated into red blood cells in vivo Nat.Biotechnol. 20: 467-472; 2002). In addition, in vitro production of erythrocytes induced in human pluripotent stem (hiPS) cells has been reported (Lapillonne, H .; Kobari, L .; Mazurier, C .; Tropel, P .; Giarratana). , MC; Zanella-Cleon, I .; Kiger, L .; Wattenhofer-Donze, M .; Puccio, H .; Hebert, N .; Francina, A .; Andreu, G .; Viville, S .; Douay, L Red blood cells generation from human induced pluripotent stem cells: perspectives for transfusion medicine.Haematologica; 2010. [Epub ahead of print]).

However, the resulting erythroblasts show timely differentiation progression, inadequate denucleation and greater vulnerability, which impairs final erythrocyte differentiation in these artificial systems (Choong, ML; Yang, HH; McNiece, I). MicroRNA expression profiling during human cord blood-derived CD34 cell erythropoiesis.Exp. Hematol. 35: 551-564; 2007; Ronzoni, L .; Bonara, P .; Rusconi, D .; Frugoni, C .; Libani, I. ; Cappellini, MD Erythroid differentiation and maturation from peripheral CD34 + cells in liquid culture: cellular and molecular characterization. Blood Cells Mol. Dis. 40: 148-155; 2008). Erythropoiesis is a complex and multistep process. Hematopoietic stem cells (HSCs) differentiate into erythroid progenitors and then into mature erythrocytes. Denuclearization is the last step in this process. However, the understanding of the molecular and cellular mechanisms that underlie this process is insufficient.

In addition, other previous studies on in vitro red blood cell production reported defects in cell morphology, limited proliferation, and low denucleation rate, despite the addition of serum (10-30%). Moreover, no reports describe the storage of extracellularly produced red blood cells. Survival of erythrocytes produced in vitro is only a few days, and the survival rate is known to be very poor (Baek, EJ; Kim, HS; Kim, JH; Kim, NJ; Kim, HO Stroma-free mass production of clinical-grade). red blood cells (RBCs) by using poloxamer 188 as an RBC survival enhancer.Transfusion 49: 2285-2295; 2009). Earlier optimism that artificially produced red blood cells would soon emerge was not realized. There have been no reports of mass production of viable denuclear erythrocytes in the absence of stromal cells and serum and plasma (Leberbauer, C .; Boulme, F .; Unfried, G .; Huber, J .; Beug , H .; Mullner, EW Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors.Blood 105: 85-94; 2005; Narayan, AD; Ersek, A .; Campbell, TA; Colon, DM ; Pixley, JS; Zanjani, ED The effect of hypoxia and stem cell source on haemoglobin switching.Br. J. Haematol. 128: 562-570; 2005). Therefore, the development of safe and efficient culture systems and storage methods for the production of red blood cells without supplementation of additional additives such as serum and stromal cells remains an important research task.

The inventors have overcome the problems of the prior art for mass production and storage of clinical grade red blood cells, and by using a simple method of temperature control without the use of serum, plasma, and stromal cells, healthy in vitro production red blood cells (in vitro) It was confirmed that a large amount of genertated RBC, cRBC) (artificial erythrocytes) can be obtained, and a method of effectively storing the same has been completed.

The main object of the present invention is to provide an in vitro mass production method of erythrocytes, capable of mass production of clinical grade erythrocytes while overcoming the problems of the prior art described above.

Another object of the present invention is to provide an in vitro mass production medium of red blood cells, which is used for mass production of red blood cells.

It is to provide a method for storing artificial red blood cells, which can effectively store the artificial red blood cells obtained by the method of the present invention.

In order to solve the above problems, the present invention is an aspect,

In vitro mass production of erythrocytes, comprising culturing erythrocyte progenitor cells in a stroma-free, serum-free and plasma-free medium, including an energy source and antioxidants Method and;

It provides a medium for mass production of erythrocytes in vitro comprising the energy source and antioxidants, characterized by a stroma-free, serum-free and plasma-free.

At this time, the antioxidant is preferably vitamin C.

The erythrocyte progenitor cells may be selected from the group consisting of progenitor cells, basophilic erythrocytes, polyinflammatory erythrocytes, and sperm erythrocytes.

The medium may be a cell culture minimum medium (CCMM) containing only a carbon source, a nitrogen source and a trace element component, wherein the carbon source serves as an energy source. That is, as sources of energy, polysaccharides, monosaccharides, organic acids, amino acids, alcohols, alcohols, polycyclic compounds, cellulose, hemicellulose Various carbon sources such as hemocellurose and lignin can be used. Examples of the polysaccharide include dextrin and starch, and examples of the disaccharide include sucrose, maltose, trehalose, and mannitol. Examples of the monosaccharide include glucose, mannose, fructose, and galactose. Can be mentioned.

As the cell culture minimal medium, DMEM (Dulbecco's Modified Eagle's Medium), EDM (Endothelial differentiation medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium.

On the other hand, the medium of the present invention, if necessary, stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony It may further include a combination of one or more of the stimulation factor or erythropoietin.

The culture can be incubated for a suitable period of time based on ordinary skill in the art, for example, can be cultured for a period of about 25 days.

At this time, it is preferable to incubate at a low temperature of 25 ~ 27 ℃ in the second half of the culture period. That is, in one embodiment, the low temperature culture may be made 17 days after the start of the culture.

On the other hand, the present invention is another aspect,

Provides an in vitro mass production method of red blood cells, including:

In the early differentiation phase of erythrocyte progenitor cells, culturing erythrocyte progenitor cells in a stroma-free, serum-free and plasma-free medium containing an energy source and antioxidant at room temperature. ; And

In the late differentiation phase of erythrocyte progenitor cells, cultured at low temperature conditions of 25-27 ° C. in mineral-free, serum-free and plasma-free medium containing an energy source and antioxidants. Steps.

The related description is the same as described above.

In another aspect, the present invention,

It provides a method for storing artificial red blood cells, characterized in that by adding the plasma to the artificial red blood cells by the above method. Preferably, the plasma is used that is derived from umbilical cord blood.

Obtaining red blood cells in accordance with the present invention provides the nutrition and environment necessary for the differentiation, proliferation and maintenance of cells without coculture with supporting stromal cells and without the addition of serum and plasma, resulting in highly efficient nuclear outflow ( By inducing enuleation, erythrocyte cells can be mass produced.

Therefore, the present invention does not include many cell lines used in co-culture, foreign factors such as fetal bovine serum (FBS), which are problematic for safety, and human plasma, serum, and stromal cells that are expensive, difficult to obtain, and difficult to guarantee safety. By culturing with the medium, erythrocyte progenitor cells can be differentiated purely into erythroid cells and proliferated in large quantities, as well as denuclearization, thereby producing high quality red blood cells. And above all, the simplicity and low-cost economics of the method of the present invention are also very advantageous, which is a very important factor in the industrial mass production of red blood cells.

In addition, there have been no examples of long-term storage of artificial red blood cells, but the artificial red blood cells obtained by the present invention can be stored for a long period of time by being stored in a solution containing plasma, which is more useful for clinical use.

1 is a graph showing amplification and cumulative amplification multipliers of erythroid cells up to medium exchange time in cells divided and divided into various conditions, and differentiation results into red blood cells with or without vitamin C. FIG.
Figure 2 is a result of confirming the differentiation into erythrocytes with or without SR and serum according to an embodiment of the present invention by morphological changes, markers, denucleation rate, etc. by date.
Figure 3 is the result of measuring the hematological parameters of the cultured red blood cells.
Figure 4 is a result confirming the shelf life of the cultured red blood cells at 4 ℃.
5 is a result of observing cell viability, cell motility, denucleation rate, morphology, etc. to investigate the effect of hypothermia on the final maturation of red blood cells.
Figure 6 shows the results of observing osmolarity fragility to investigate the effect of low temperature on the final maturation of red blood cells.
7 is a photomicrograph of the case where plasma is used in the conventional artificial red blood cell production.

Definitions of terms used in the present invention are as follows.

'Progenitor cells' are undifferentiated cells with self-replicating ability and differentiation potency, but are ultimately differentiated cells in which the type of cells to differentiate is already determined. Progenitor cells have a predetermined differentiation pathway, but generally do not express markers of mature fully differentiated cells or function as mature fully differentiated cells. Thus, progenitor cells differentiate into related cell types but cannot form a wide variety of cell types under normal conditions. In the present invention, red blood cell progenitor cells are used.

The term 'culture' refers to the growth of organisms (mainly microorganisms and embryos of living plants and animals) or parts of organisms (organs, tissues, cells, etc.) under appropriate artificial control of environmental conditions. In this case, temperature, humidity, light, and gas phase composition (partial pressure of carbon dioxide and oxygen) are important as external conditions, and the most important direct influence on other cultured organisms is medium.

"Medium" refers to a mixture for growth and proliferation of cells and the like in vitro, including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals and the like. In particular, the medium of the present invention is a medium for obtaining a large amount of red blood cells through erythrocyte progenitor cell culture.

'Serum' is a supernatant obtained by centrifugation from the blood of an animal or human. The serum includes trace elements including various factors such as various inorganic salts, polypeptide growth factors, and polypeptide hormones, which are essential for growth but not clearly identified, in addition to essential nutrients required for cell growth.

'Differentiation' refers to a phenomenon in which structures or functions are specialized during the division and proliferation of cells, that is, the shape or function of a living cell or tissue is changed to perform a given task. Generally, a relatively simple system is divided into two or more qualitatively different sub systems. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons. Phosphorus difference, or as a result, is a state divided into subclasses or subclasses that can be distinguished qualitatively.

In vitro large-scale production refers to the isolation and culturing of erythrocyte progenitor cells to yield high levels of erythrocytes that may be clinically useful. In the present invention, 'mass production' means that 'proliferation' or 'expansion' or 'expansion' of denucleated erythrocytes occurs sufficiently due to the effective differentiation of erythrocyte progenitor cells.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

Hereinafter, the present invention will be described in detail.

The present inventors endeavored to develop a method for mass production of red blood cells in vitro, and as a result, when culturing the cells in the absence of serum, plasma and feeder cells (substrate) at the time of culturing erythrocyte progenitor cells, We solved the previous problem described above and found that viable red blood cells can be mass produced in vitro to be clinically useful.

The present invention is the first invention to initiate mass production and long term storage of denuclear red blood cells in a medium free of serum, plasma and feeder cells (substrate).

In one aspect, the invention includes culturing red blood cell progenitor cells in a stroma-free, serum-free and plasma-free medium, including an energy source and an antioxidant, An in vitro mass production method of red blood cells and an in vitro mass production medium of red blood cells used therein.

In the present specification, the erythroid progenitor cell refers to all cells included in the erythrocyte formation process except for the erythrocytes in which the maturation process is completed.

The erythrocyte progenitor cells differentiate into mature erythrocytes through erythropoiesis consisting of the following steps: (a) from unipotent stem cells (hemocytoblast) to proerythroblasts; Differentiating; (b) differentiating from progenitor cells to basophilic erythroblasts; (c) differentiating from basophilic erythrocytes to polychromatophilic erythroblasts; (d) differentiating from multibasic erythrocytes to orthochromatic erythroblasts; (e) differentiating into polychromatic erythrocytes in infectious erythrocytes; And (f) differentiating from erythrocytes to erythrocytes.

Thus, the erythroid progenitor cells may be pro-blasts, basophilic erythrocytes, polyinflammatory erythrocytes or spermatogenic erythrocytes. In addition, they may be derived from all cell source-embryonic stem cells capable of differentiating into erythroid progenitor cells, iPS cells (induced pluripotent stem cells), adipose derived hematopoietic stem cells, and the like.

The erythrocyte progenitor cells may be characterized as being CD34 + cells.

CD34 + cells refer to cells that express CD34. CD34 is the name of the human gene encoding the protein. The CD34 molecule is a glycoprotein on the cell surface, which is a differentiation molecule present in specific cells in the human body, and forms a group and functions as a cell-cell adhesion factor. It also mediates the contact of stem cells to extracellular matrix or stromal cells of the bone marrow. Cells expressing CD34 are found in umbilical cords and bone marrow, such as hematopoietic cells of normal blood vessels, endothelial progenitor cells, and endothelial cells, but are not limited thereto.

Erythrocyte progenitor cells can be obtained from a variety of sources, such as peripheral blood, umbilical cord blood, or bone marrow. According to a preferred embodiment of the invention the CD34 + cells are cells derived from umbilical cord blood. CD34 + cells as erythroid progenitors can be isolated according to various cell isolation methods known in the art, such as immunomagnetic-bead isolation methods using CD34 + antibodies.

In addition, the medium used in the present invention may be a cell culture minimum medium (CCMM) containing only a carbon source, a nitrogen source and a trace element component as a medium for obtaining red blood cells from red blood cell progenitor cells in vitro.

That is, it is a mixture containing essential sugars, amino acids, water, etc. necessary for the cells to live, and is a basic medium excluding serum, nutritional substances, and various growth factors.

In particular, as a source of energy, polysaccharides, monosaccharides, organic acids, amino acids, alcohols, alcohols, polycyclic compounds, cellulose, hemicellulose Various carbon sources such as hemocellurose and lignin are used. Examples of the polysaccharide include dextrin and starch, and examples of the disaccharide include sucrose, maltose, trehalose, and mannitol. Examples of the monosaccharide include glucose, mannose, fructose, and galactose. Can be mentioned.

The cell culture minimum medium (CCMM) of the present invention may be prepared by artificially synthesizing or using a commercially prepared medium. Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited thereto.

A key feature of the medium for obtaining erythrocytes from erythrocyte progenitor cells in vitro of the present invention is the inclusion of antioxidants.

The antioxidants include, but are not limited to, natural antioxidants Tocopherol, Sesamol, Gingeron, Capsaicin, Quercetin, Gallic acid; Synthetic antioxidants BHA, BHT, TBHQ; Citric acid, ascorbic acid, an effect-boosting agent, can be selected and used as a water-soluble antioxidant vitamin C, a fat-soluble antioxidant vitamin E, ubiquinone (CoQ), carotenoids, etc., and most preferably vitamin C do.

The addition of vitamin C can result in red blood cells with a much higher cell viability. The vitamin C is because the red blood cell progenitor cells can maintain a stable state from oxidative stress in vitro.

Another feature of the present invention is that it is a "stroma-free, serum-free and plasma-free medium."

"Stroma-free, serum-free, and plasma-free" means the absence or substantially culture of both feeder stromal cells, serum, and plasma; It is meant to include a small amount so as not to exert its functions in the phase.

Previous studies of in vitro red blood cell production have generally used conditions involving serum or fetal bovine serum (FBS).

Serum is derived from blood, including a wide range of macromolecules, carrier proteins for lipoid and trace elements, cell attachment and spreading factors, low molecular weight nutrients, and hormones and growth factors. Refers to the fraction derived. Operationally, serum can be defined as the proteinaceous, acellular fraction of blood that remains after removal of the aggregation components of red blood cells, platelets and plasma. The most widely used animal serum for cell culture is fetal bovine serum, FBS, but adult bovine serum, horse serum and protein fractions thereof (eg Fraction V serum albumin) have been used. However, the cost is high, and proteins and peptides contained in serum make the purification process of the product obtained after cell culture very complicated. In addition, the sterilization of serum increases the possibility of contamination by mycoplasma or virus because it must be filtered without heating. Serum-containing media also bubbles well. However, the biggest problem with the use of serum is that it is difficult to obtain reproducibility because the composition of serum varies from batch to batch. Serum-free medium has been developed to overcome this problem.

For serum-free culture of erythrocyte cells, feeder stromal cells were used to provide a microenvironment (Ma, F .; Ebihara, Y .; Umeda, K .; Sakai, H .; Hanada , S .; Zhang, H .; Zaike, Y .; Tsuchida, E .; Nakahata, T .; Nakauchi, H .; Tsuji, K. Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis.Proc.Natl Acad.Sci. USA 105: 13087-13092; 2008; Olivier, EN; Qiu, C .; Velho, M .; Hirsch, RE; Bouhassira, EE Large-scale production of embryonic red blood cells from human embryonic stem cells. Exp.Hematol. 34: 1635-1642; 2006; Qiu, C .; Olivier, EN; Velho, M .; Bouhassira, EE Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells 111: 2400-2408; 2008).

Also, except for this, most of the cells are known to die except for plasma, and even when plasma is added as shown in FIG. 7, healthy red blood cells could not be produced by the conventional method.

Thus, it is known that it is not possible to produce enough red blood cells for transfusion processes that require large amounts of media and cells without removing all serum, plasma and stromal cells (Timmins, NE; Nielsen, LK Blood cell manufacture: current methods and future challenges.Trends Biotechnol. 27: 415-422; 2009).

However, the present invention confirmed that the use of a medium free of serum, plasma, and feeder cells, including essential antioxidants such as vitamin C, was more effective for the production of healthy red blood cells, thereby improving red blood cells having improved integrity and viability. Provide in vitro production.

Thus, in one embodiment the present invention

A red blood cell in vitro mass production medium comprising an energy source and vitamin C, characterized by a stroma-free, serum-free and plasma-free.

In another embodiment the invention comprises culturing red blood cell progenitor cells in a stroma-free, serum-free and plasma-free medium comprising an energy source and vitamin C. The present invention relates to an in vitro mass production method of red blood cells.

The medium may further include antibiotics such as penicillin, streptomycin, or gentamicin, as necessary, and may be used to control the proliferation and differentiation of erythrocyte progenitor cells, interleukin-1. May further comprise at least one of interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor or erythropoietin. .

The present invention also relates to an in vitro mass production method of erythrocytes, which is mainly characterized by different temperature conditions for a certain period of time.

In particular, such culture temperature conditions also become a factor that can significantly improve the viability of the cells.

Erythrocytes become red blood cells by passing the nucleus at the last differentiation stage, greatly altering the internal structure, and the loss of internal organelles. However, in vitro conditions, the viability of erythroid cells drops significantly at this last differentiation time. Erythrocyte cells prepared according to most prior studies are damaged cells that only survive for a few days, and their viability is so low that they are virtually unavailable for transfusion. Therefore, the existing research on erythropoiesis could not be conducted properly.

In the present invention, however, the viability of erythrocytes is incubated by incubating the culture temperature conditions at a low temperature of 25 to 27 ° C., preferably at 27 ° C., from 17 days after the last differentiation step, for example, 17 days after the start of the culture. To maintain cell motility and maturity. In addition, the denuclearization rate also increases significantly.

This is because the activity of various enzymes involved in cell death is inhibited at 27 ° C.

Therefore, the present invention is in one embodiment,

In an early differentiation phase of erythrocyte progenitor cells, culturing the erythrocyte progenitor cells in a stroma-free, serum-free and plasma-free medium containing an energy source; And

In the late differentiation phase of erythrocyte progenitor cells, the step of incubating at low temperature conditions of 25-27 ° C. in mineral, serum-free and plasma-free medium containing an energy source is performed. It provides, in vitro, mass production of red blood cells.

The related description is as described above.

Although the total duration of the erythrocyte progenitor cell culture of the present invention is not limited, for example, it may be preferably about 25 days, wherein the initial differentiation period of the erythrocyte progenitor cells may be a period of about 17 days after the start of the culture, The late differentiator of the erythroid progenitor cells may be a period of about 17 to 25 days after the start of the culture. It will be apparent that addition and subtraction are possible depending on the needs of those skilled in the art.

The present invention provides a culture environment of erythrocyte precursors in conditions of serum-free, plasma-free, mineral conditions and / or temperature change according to differentiation time, thereby increasing cell viability of erythrocyte precursors and obtaining denuclearized erythrocytes with high efficiency. do. In addition, more red blood cells can be obtained by adding antioxidants such as vitamin C.

Thus, the method according to the present invention and the medium used therein may be applied to the "mass production" of human erythrocytes through bioreactor systems and achieve clinically useful levels.

In another aspect, the present invention relates to a long-term storage method of artificial red blood cells obtained by the above method.

To date, no successful cases of artificial red blood cell storage for a month have been reported.

In the present invention, however, plasma was added to the artificial red blood cells obtained by the above method and refrigerated at 4 ° C. for 4 weeks, that is, about 1 month of long-term storage. At this time, the origin of plasma used is not limited, but for example, those derived from umbilical cord blood can be used.

On the other hand, red blood cells obtained by the method of the present invention may have various therapeutic applications.

Moreover, the increased number of erythrocytes induced by the present invention can be used in novel therapeutic strategies in the treatment of hematopoietic disorders, or in blood banks.

In certain embodiments, the applied red blood cells can be used for transfusion. The ability to produce large amounts of cells for transfusion can alleviate the chronic shortage of blood experienced in blood banks and hospitals nationwide.

Certain aspects of the present invention relate to increasing human erythrocytes to reach commercial amounts and to prolonged storage of such artificial human erythrocytes.

A method for producing, storing, and distributing erythrocyte cells expanded by the methods described herein. After producing and expanding human erythrocytes in vitro, human erythrocytes can be harvested, purified and optionally stored prior to treatment of the patient. Thus, in a particular embodiment, the present invention provides a method of supplying red blood cells to hospitals, health care centers, and clinicians, whereby the artificial red blood cells produced by the method described herein are stored and stored in a hospital. It may be ordered and administered to patients in need of erythrocyte cell therapy, as required by a healthcare center, or clinician.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: Production of red blood cells and confirmation of various characteristics according to the present invention

1. Cell culture and yield enumeration )

CD34 + was isolated from cord blood of healthy donors using an immunomagnetic microbead selection method and the EasySep CD34 Separation Kit (StemCell Technologies, Vancouver, Canada).

The isolated CD34 + cells were cultured at 1 × 10 ⁵ cells / ml for 21-26 days in mineral (stroma-free) conditions. Isolve's modified Dulbecco's medium (Gibco, Grand Island, NY) or Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, Mo.) at 150 μg / ml Sigma-Aldrich), 160 μM monothioglycerol (Sigma-Aldrich), 2 μg / ml cholesterol (Sigma-Aldrich), lipid mixture (Sigma-Aldrich), and 1% penicillin-streptomycin solution (Gibco) Added. In some experiments 30.8 μM of L-ascorbic acid (Sigma-Aldrich) was added. In the experiment using IMDM, 1% of human albumin (Green Cross, Korea) was added.

Several cytokines were added to differentiate CD34 + cells.

From day 0 to day 7, 1 × 10 μM hydrocortisone (Sigma), 100 ng / ml stem cell factor (SCF; R & D systems, Minneapolis, MN, USA), 10 ng / ml interleukin ( 6-well or 12- in medium supplemented with IL) -3 (R & D systems, Minneapolis, MN, USA) and 6 IU / ml of erythropoietin (EPO; Calbiochem, La Jolla, CA, USA) The cells were cultured in well plates (BD Falcon, Franklin Lakes, NJ). From day 7 to day 13, proliferated erythroblasts were cultured in the presence of 50 ng / ml SCF, 10 ng / ml IL-3 and 3 IU / ml EPO. From day 13 to day 17, 50 ng / ml SCF and 2 IU / ml EPO were added.

From day 13 the culture medium was divided into four different conditions: ± 10% FBS (TerraCell International, Ontario, Canada) or ± 10% Serum replacement reagent (SR; Gibco). The culture medium was changed every 2-3 days and incubated in a 37 ° C. CO ₂ incubator (Sanyo, Japan).

Trypan blue staining was used for all cell counts, and only live cells were included in the fold expansion results.

2. Intracellular  activation Of reactive oxygen species (ROS)  Measure

The production of intracellular ROS was measured using oxidation sensitive 2,7-dichlorodihydrofluorescein diacetate (DCF-DA, Molecular Probes, Eugene, OR). A total of 1 × 10 5 cells from each step were pre-washed and resuspended in heated phosphate buffered saline (PBS). 200 M DCF-DA was added to the cells and incubated in the dark at 37 ° C. for 15 minutes in a 5% carbon dioxide incubator. Fluorescence intensity by oxidative conversion of DCF-DA was immediately measured using flow cytometry.

3. Imaging of Red Blood Cells

Cell morphology was analyzed by cytospin method using cell centrifuge (Cellspin, Hanil Science Industrial, Seoul, Korea) followed by Wright-Giemsa staining (Sigma-Aldrich). Pictures of the stained cells were taken with a digital camera (Eclipse TE2000-U, Nikon, Japan) and used the Nikon ACT-1 program.

4. Of red blood cell detection Flow cytometry  analysis

To measure phenotypic changes during red blood cell growth, the following anti-human antibodies were attached to the cells on days 13, 17 and 21: (i) Glycoporin A (GPA) -FITC (BD PharMingen, Franklin Lakes, NJ). ) (ii) CD71-FITC (eBioscience, San Diego, CA). Immunoglobulin G1 (IgG1) -FITC and IgG1-PE (Beckman Coulter, Miami, FL) were used for homogeneous control staining. For immunophenotype, cells were washed in phosphate buffered saline (PBS) and incubated with each monoclonal antibody for 15 min at 4 ° C. After washing, the cells were suspended in phosphate buffered saline (PBS) and analyzed using a flow cytometer.

Intracellular expression of hemoglobin (Hb) was analyzed using FITC conjugated anti-fetal hemoglobin (HbF) and PerCP conjugated anti-dollar hemoglobin (Hbβ, BD PharMingen). Briefly, cells are resuspended in cold 0.05% glutaraldehyde for 10 minutes at room temperature, washed in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin, and at room temperature. It was suspended for 5 minutes in 0.1% Triton X-100. After washing, antibody was added to the cells for 15 minutes at room temperature and washed. Stained cells were analyzed using flow cytometer BD FACSCalibur ™ (BD Biosciences, San Jose, Calif.) And provided software.

5. RT - PCR

Total RNA was isolated using Trizol (Invitrogen) and reverse transcribed the total RNA using SuperScript III reverse transcription enzyme (Invitrogen). RT-PCR was performed on triplicates (LightCycler 480 II; Roche Diagnostics) using SYBR Premix ExTaq (Takara-Bio, Shiga, Japan). Relative quantitation was achieved by normalization to endogenous β-actin.

The sequence of the primer set used is as follows (forward / reverse):

β-globin: 5'-GAAGGCTCATGGCAAGAAAG-3 '(SEQ ID NO: 1)

           5'-CACTGGTGGGGTGAATTCTT-3 '(SEQ ID NO: 2);

γ-globin: 5'-GCTGACTTCCTTGGGAGATG-3 '(SEQ ID NO: 3)

           5'-GAATTCTTTGCCGAAATGGA-3 '(SEQ ID NO: 4);

GATA1: 5'-CCAAGCTTCGTGGAACTCTC-3 '(SEQ ID NO: 5)

5'-CCTGCCCGTTTACTGACAAT-3 '(SEQ ID NO: 6).

6. of manufactured red blood cells ABO Mold and Rh Slide inspection for mold determination

The ABO group and Rh expression of red blood cells (cRBCs) (artificial red blood cells) cultured in vitro were examined and compared with the donor's original red blood cells. One drop of cells was placed on a glass slide and one drop of anti-A, anti-B, anti-C, and anti-D reagents were dropped. The slide was tilted slowly and the aggregation was observed.

7. Evaluation of Hematological Parameters

Cultured cells were collected on day 21 and Sysmex XE-2100 hematology analyzer (Sysmex, Kobe, Japan) to compare blood cell parameters, including hemoglobin levels and mean hemoglobin (MCH), with donated peripheral red blood cells. Measured using.

8. Cultured red blood cells ( cRBCs Measurement of oxygen transport capacity

To demonstrate the oxygen transport capacity of cultured red blood cells (cRBCs), the saturation of oxygen (sulfur dioxide (SO 2)) was calculated. Based on the older spectroscopy calculation of oxygen saturation, Refsum, HE; Sveinsson, SL Spectrophotometric determination of hemoglobin oxygen saturation in hemolyzed whole blood.Scand. J. Clin. Lab. Invest. 8: 67-70; 1956 Corrected. In summary, the absorbance was measured using a spectrophotometer at 560 nm and 576 nm where the absorbance of HbO2 was the maximum and minimum. These measurements, constants, and equations of HE Refsum and SL Sveinsson were used to calculate SO2 values for peripheral blood, CB, and cultured red blood cells (cRBCs) (Refsum, HE; Sveinsson, SL Spectrophotometric determination of hemoglobin oxygen saturation in hemolyzed whole blood.Scand. J. Clin.Lab.Invest. 8: 67-70; 1956). Low oxygen partial pressure was achieved by incubation in a low oxygen incubator (Sanyo) of 1.5% for 12 hours. The partial pressure of oxygen was measured at a critical point near 50 mmHg, the peripheral blood pressure with the largest SO2 difference between fetal and adult hemoglobin. However, the technical limitations of using a low-oxygen incubator rather than an autoanalyzer did not map the entire range of 0-120.

9. Red blood cells Strain ( deformability ) Measure

In order to withstand the pressure of various blood flows in the human body, and to measure whether there is a deformability of the cell membrane in capillaries, etc., it was tested using a device capable of sensitive measurement (Rheoscan (Rheomeditech, Seoul, Korea)) (Biorheology 2009; 46: 251-264).

10. Cell Viable  Measure

Cell viability was measured by flow cytometry using trypan blue staining and Annexin V analysis. Cells were stained for 15 minutes at room temperature with Annexin V-PE and propidium iodide (PI, Invitrogen, Camarillo, Calif.). The cells were washed twice with wash buffer and analyzed by flow cytometry.

11. Chemical Analysis

The pH, calcium ion (Ca2 +) and potassium ion (K +) concentrations of the medium used to culture red blood cells for 18 to 21 days were analyzed using RapidSystems' blood gas analyzer (Siemens, USA).

12. Statistical Analysis

Statistical analysis was performed using Prism software (Prism, Version 5.0, GraphPad, San Diego, CA). Results are expressed as means ± standard error of mean (sem). Statistical significance was set at p values of 0.05 or less. Paired t-tests were used to compare final cell numbers under ± FBS conditions and two temperature effects. Final cell products under ± SR and ± FBS conditions were compared by unidirectional ANOVA.

Example  2: test result of red blood cell characteristics according to the present invention

(1) Effect of Serum and Substrate-Free Culture Conditions on Erythrocyte Cell Acquisition

In previous results reports, extracellular red blood cell production was performed under conditions involving plasma or serum.

In this study, however, cultures without plasma or serum were established. Since cell death was expected to be increased under these conditions, the effects of serum replacement (SR) reagents that were added to support and nourish the growth of various stem cells in general instead of FBS were measured. To assess the effect of serum and substrate-free culture conditions, CD34 + HSCs were isolated from CB (purity> 96% of each) incubated for 21 days. CD34 + cells were cultured in erythrocyte differentiation medium including EPO, IL-3, and SCF. On day 13 of culture, cells were isolated in medium under different conditions, with or without serum. On day 0 of culture, addition of EPO, IL-3, and SCF resulted in cell proliferation with an average of 30,000-fold amplification compared to the initial cell number after 13 days.

Final cell count is one of the most important factors for clinical application of the present invention, so we compared the cell count on day 21. Final proliferation fold of red blood cells from day 13 to 21 was 2.1 than in serum free conditions (+ FBS, 6.4-fold; -FBS, 13.4-fold) (paired t-test, p <0.05, n = 10) (FIG. 1A). It was twice as big. The two groups were then subdivided into four groups divided by medium with or without SR composition. On the 21st day, the + SR-FBS condition (16.3 times) showed 5.3 times higher proliferation than the -SR-FBS condition (11.0 times). The subgroups showed no significant difference, demonstrating little effect without SR under serum-free conditions (unidirectional ANOVA, p> 0.05) (FIG. 1B).

Therefore, the inventors were able to obtain more than ⁵ × 10 ⁵ times from one CD34 + cell in the absence of serum and SR. This theoretically confirmed that it was possible to obtain 1x10 ¹² erythrocytes when experimenting with whole CD34 + cells (2x10 ⁶ cells) in one CB.

That is, according to the results of counting only living cells, the final erythrocyte product in serum-free medium obtained 2.1 times more significant difference (p <0.05, n = 10) (FIG. 1A, B), and each condition was SR. In comparison with the more granularity, the addition of SR after 19 days of cell damage was found to be more helpful for cell harvesting, and the serum-free condition was better than that with serum.

(2) the effect of vitamin C

The present inventors attempted to determine whether vitamin C remains stable in vitro from oxidative stress.

Apoptosis was reduced by 10.8% at day 21 of culture in the presence of vitamin C compared to the control condition (Fig. 1C). Without vitamin C, the recovery of final product was poor on day 21 of cultivation, with little viable cells. On the other hand, when vitamin C was added, red blood cells that were not easily broken could be obtained. The average level of ROS was also reduced by 12% (FIG. 1D), which means that vitamin C further stabilizes cells from oxidative stress in serum-free, mineral conditions.

Therefore, all of the following serum-free experiments were conducted by adding vitamin C at the same concentration.

(3) differentiation of human red blood cells

Cell growth and differentiation were detected by Wright-Giemsa staining.

At each culture step, the appearance of erythrocytes and erythrocytes was similar, with or without serum or SR, and suitable erythrocytes were produced. At 14 days of culture, basophilic erythroblasts were observed in which the cells were large in size, large in nucleus, and bluish in color.

In previous studies, the most critical limit for erythrocyte production under serum and substrate-free conditions was low nuclear clearance (Freyssinier, JM; Lecoq-Lafon, C .; Amsellem, S .; Picard, F .; Ducrocq, R .; Mayeux, P .; Lacombe, C .; Fichelson, S. Purification, amplification and characterization of a population of human erythroid progenitors.Br. J. Haematol. 106: 912-922; 1999; Malik, P .; Fisher, TC; Barsky, LL; Zeng, L .; Izadi, P .; Hiti, AL; Weinberg, KI; Coates, TD; Meiselman, HJ; Kohn, DB An in vitro model of human red blood cell production from hematopoietic progenitor cells.Blood 91: 2664 -2671; 1998; Neildez-Nguyen, TM; Wajcman, H .; Marden, MC; Bensidhoum, M .; Moncollin, V .; Giarratana, MC; Kobari, L .; Thierry, D .; Douay, L. Human erythroid cells produced ex vivo at large scale differentiated into red blood cells in vivo.Nat.Biotechnol. 20: 467-472; 2002).

As a result of cell differentiation into orthochromic erythroblasts, the average cell size decreased over time after 15 days. On the 18th day of culture, most of the cells were orthochromic erythroblasts, which are pre-denuclear erythrocytes. From day 19 more denuclear red blood cells appeared in serum-free medium than in serum-containing medium. Red blood cells began to be produced on day 21 of the culture, and there was a large difference in their maturation time and degree of denuclearization between cells from different individuals (FIG. 2A). Therefore, some cells continued to denuclearize even after 25 days of culture for denuclearization. On day 22 of the culture, cells cultured without SR and FBS were filtered with two leukocyte removal filters to obtain high-purity red blood cells (indicated by arrows in FIG. 2A are denucleated red blood cells).

When comparing the denuclearization rate on day 21, the denucleation rate was measured by 3.74 times higher in cells cultured without serum than cells cultured with serum. The denucleation rate could not be determined because most of the cells died in some cases involving serum. Although the rate of denucleation was relatively low at 37 ° C., the present invention clearly showed an increase in the rate of denucleation under conditions without serum (p <0.005).

Cell surface markers transferrin receptor (CD71) / glycophorin A (GPA) (both markers for mature erythrocytes) to investigate whether final red blood cell production proceeds correctly in the absence of serum and substrate. And final erythropoiesis was also measured using FACS analysis based on adult / fetal hemoglobin (Hbβ and Hbγ) expression profiles. More than 95% of the cells expressed CD71 / GPA and Hbβ / Hbγ in the absence of serum and stroma (FIG. 2B).

The maturation of erythrocyte cells was also achieved by upregulation of erythrocyte-specific gene β- and γ-globin, GATA1 transcription factor expression through the erythrocyte differentiation step (FIG. 2C). And maturation of red blood cells was confirmed by Western blot analysis of band3. Increased cell surface expression of band3 was seen from the erythrocyte stage to the reticulocyte stage (FIG. 2D). These data correlate with maturation stages measured with morphological changes as shown in FIG. 2A.

In addition, in the late stage (20-22 days), more than 90% of cells cultured in serum-free medium still survived, whereas only 60-80% of cells cultured in the presence of serum survived (FIG. 2E).

(4) Hematological parameters and functional analysis of cultured red blood cells

On day 21, the culture final product was purified by passing through a leukocyte removal filter to remove erythrocytes and nucleated erythrocytes (FIG. 3A). The blood type of cultured erythrocytes corresponded to the blood type of donated erythrocytes (FIG. 3B). The hematological parameters of cultured erythrocytes were similar to those of donated original erythrocytes (Hb 12.29 ± 2.91 g / dL; mean cell volume, 99.72 ± 0.58 fL; and mean cell hemoglobin, 27.31 ± 6.47 pg, n = 3) .

In addition, the present inventors measured the function of hemoglobin according to oxygen carrying capacity using a low oxygen incubator and a gas analyzer (RAPID Lab 1265). The calculated oxygen saturation (SO ₂ ) values were CB RBC (63.6%)> cRBCs (59.4 ± 2.1%)> PB RBC (54.6 ± 5.6%). We could expect that cRBCs would function normally because the results were similar to normal blood controls. G6PD (glucose-6-phosphate dehydrogenase) activity, which decreases glutathione and maintains ATP levels, is 515.25 mU / ml, and the appropriate modifying capacity of cRBC is shown in comparison with peripheral blood natural RBCs (PB RBCs) (FIG. 3). C and D).

(5) cold conditions in the final maturation of red blood cells ( hypothermia ) Influence

Cell viability was reduced from day 17, and the effect of hypothermia on the maturation of red blood cells was investigated to inhibit the disproportionate apoptosis process.

Survival of RBCs cultured at 27 ° C. increased to about 17.4% between 19 and 21 days (FIG. 5A), and not only was well preserved in form, but also maintained cell motility and maturation capacity, resulting in a significant increase in denuclearization rate. (FIG. 5B)

 In addition, these cell viability was confirmed to be significantly improved at low temperature conditions.

This is thought to be because the activity of various enzymes involved in cell death is inhibited at 27 ° C. In the mitochondrial dehydrogenase activity during apoptosis, the effector activity of cells cultured at 27 ° C. was reduced by 35% compared to the case of 37 ° C. (data not shown).

In addition, through electron microscopy, better morphology and intactness were observed at low temperatures of 27 ° C. (FIGS. 5C and D), and confirmed by osmolarity fragility (osmotic fragility) analysis (FIG. 6).

Therefore, it was found that the cell membrane became weak when the culture was maintained at 37 ° C until the end. Therefore, in the late stage of erythrocyte differentiation, culturing at a low temperature of 27 ° C. is advantageous for obtaining erythrocytes having excellent cell viability.

Example  3: Storage and storage of the obtained artificial red blood cells

In addition, the present inventors have sought to find a method of effectively storing and storing the obtained red blood cells.

To this end, it was tested whether cRBC can be effectively stored in the refrigerator, ie at 4 ° C. For natural RBCs and obtained artificial erythrocytes (in vitro genertated RBCs, cRBCs), cell survival results with or without serum or plasma addition were compared. The plasma used was derived from umbilical cord blood.

The results are shown in FIG.

Both artificial red blood cells and natural red blood cells did not survive well for 2 weeks when serum was added, but when cord blood-derived plasma was added, it was confirmed that they survived for 28 days, or about a month. The donut shape of red blood cells was maintained until the last day of storage (FIG. 4B). This is the first time that artificial red blood cells have been stored for a month.

Until now, artificial erythrocytes had a short survival time and had compatibility problems, suggesting that artificial erythrocytes can be refrigerated for one month even with artificial erythrocytes.

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Claims (18)

In vitro mass production of erythrocytes, comprising culturing erythrocyte progenitor cells in a stroma-free, serum-free and plasma-free medium, including an energy source and antioxidants Way.
The in vitro mass production method of red blood cells according to claim 1, wherein the erythrocyte progenitor cells are selected from the group consisting of whole blast cells, basophilic erythrocytes, polyinflammatory erythrocytes, and basophilic erythrocytes.
The medium of claim 1, wherein the medium is Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α Erythrocyte phosphorus, characterized in that it is a cell culture minimun medium (CCMM) selected from the group consisting of α-Minimal Essential Medium (MEM), Glass's Minimal Essential Medium (G-MEM) and Iscove's Modified Dulbecco's Medium How to mass produce bit.
4. The method of mass production of red blood cells according to claim 3, wherein the antioxidant is vitamin C.
The method of claim 1, wherein the energy source is a polysaccharide (polysaccharides), monosaccharides (monosaccharides), organic acids (organic acid), amino acids (amino acid), alcohol (alcohol), polycyclic compounds (polycyclic compounds), cerulos ( cellurose), hemicelllurose (hemicellurose) and lignin (lignin) in vitro mass production method of red blood cells, characterized in that at least one carbon source selected from the group consisting of.
According to claim 1, wherein the medium is a stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor And erythropoietin further comprising at least one selected from the group consisting of erythropoietin.
The in vitro mass production method of red blood cells according to claim 1, wherein the culturing is carried out for a period of 25 days.
The method of claim 1, wherein the method further comprises culturing at a low temperature of 25 ~ 27 ℃ late incubation period in vitro mass production method of red blood cells.
The method of claim 8, wherein the low-temperature culture is in vitro mass production method of red blood cells, characterized in that made 17 days after the start of the culture.
In the early differentiation phase of erythrocyte progenitor cells, culturing erythrocyte progenitor cells in a stroma-free, serum-free and plasma-free medium containing an energy source and antioxidant at room temperature. ; And
In the late differentiation phase of erythrocyte progenitor cells, cultured at low temperature conditions of 25-27 ° C. in mineral-free, serum-free and plasma-free medium containing an energy source and antioxidants. Including the step of, in vitro mass production of red blood cells.
The in vitro mass production method of red blood cells according to claim 10, wherein the antioxidant is vitamin C.
The in vitro mass production method of red blood cells according to claim 10, wherein the erythrocyte progenitor cells are selected from the group consisting of whole blast cells, basophilic erythrocytes, polyinflammatory erythrocytes, and basophilic erythrocytes.
The method according to claim 10, wherein the medium is a stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor And erythropoietin further comprising at least one selected from the group consisting of erythropoietin.
Erythrocyte in vitro mass production medium comprising an energy source and antioxidants, characterized by a stroma-free, serum-free and plasma-free.
The medium for red blood cell in vitro mass production according to claim 14, wherein the antioxidant is vitamin C.
The method according to claim 14, wherein the medium is a stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor And at least one selected from the group consisting of erythropoietin.
A method for storing artificial red blood cells, characterized in that plasma is added to and stored in artificial red blood cells obtained by the method of claim 1 or 10.
18. The method of claim 17, wherein the plasma is derived from umbilical cord blood.

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