Patent Application
20230323300
The present invention relates to a method for in vitro production of red blood cells.
Due to the shortage of blood for transfusion, there is an increasing demand for a technique for producing red blood cells from stem cells in vitro. In Korea, about 2.1 million units of red blood cell preparations are used annually, but when it is calculated that there are 2×1012 red blood cells per unit, about 4×1018 or more red blood cells per year are needed. In the case of the United States, there is a need for 1019 or more red blood cells. Although 80 million units of collected blood are supplied worldwide every year, it is greatly insufficient to meet the demand.
Further, since red blood cell reagents used for irregular antibody screening & identification tests in transfusion medicine are also manufactured by receiving blood supplied from blood donors, it is difficult to produce and supply the reagents.
In order to commercialize a technique for in vitro culture of red blood cells to overcome various side effects and contamination and infection of blood provided by blood donors or difficulties in supply, it should be possible to automate a process capable of exhibiting an optimal red blood cell yield suitable for mass production of red blood cells of uniform quality under constant conditions. Therefore, it is essential to establish optimal culture conditions (parameters) and develop a culture technique using a bioreactor such that consistent results can be obtained.
An object of the present invention is to provide a method for in vitro production of red blood cells.
Accordingly, the present inventors have conducted various studies to establish optimal culture conditions that enable automated mass production of red blood cells using a bioreactor. As a result, it could be confirmed that during culturing of erythroid progenitor cells, by specifying the time point of conversion from stationary culture to agitation culture based on the diameter of the erythroid cells during culture, red blood cells can be efficiently produced in vitro from erythrocyte progenitor cells regardless of a culture environment such as agitation speed and without co-culturing with supporting stromal cells. That is, the present invention provides a method for preparing red blood cells at high yield by performing stationary culture in a culture vessel in an environment with a medium composition that does not include stromal cells, and converting the erythrocyte progenitor cells to agitation culture at a specific time point.
Accordingly, the present invention provides a method for in vitro production of red blood cells, including the step of converting the erythrocyte progenitor cells into agitation culture during culturing of the erythrocyte progenitor cells.
Hereinafter, the configuration of the present invention will be described in detail.
The present invention provides a method for in vitro production of red blood cells, including the step of, during the culture of erythrocyte progenitor cells, converting the cultured cells into agitation culture when the diameter of the erythrocyte progenitor cells reaches 10 to 15 μm.
First, the method for in vitro production of red blood cells of the present invention may be performed in a culture vessel. The culture vessel refers to a culture vessel capable of controlling a culture environment such as agitation speed, temperature, dissolved oxygen (DO), or pH according to the culture mode of cells, that is, stationary culture and agitation culture.
In the present invention, “progenitor cells” refers to undifferentiated cells having self-renewal ability and differentiation potency, but ultimately differentiated cells whose types of finally differentiated cells have already been determined. Although progenitor cells are committed to a differentiation pathway, they generally do not express markers of mature, fully differentiated cells or function as mature, fully differentiated cells. Therefore, although progenitor cells differentiate into related cell types, they cannot form a wide variety of cell types in a normal state. In the present invention, erythrocyte progenitor cells are used.
As used herein, “differentiation” refers to a phenomenon in which a structure or function is specialized while cells divide, proliferate, and grow, that is, means that the morphology or function of cells, tissues, and the like of an organism changes in order to carry out the tasks given to them. In general, differentiation is a phenomenon in which a relatively simple system is separated into two or more qualitatively distinct subsystems. For example, as in the distinction of the head and body between the egg parts that were initially homogeneous in ontogeny, or the distinction of cells such as muscle cells and nerve cells even in cells, a state of the development of qualitative differences between initially nearly homogeneous parts of a biological system, or the resulting separation into qualitatively distinct regions or subsystems is called differentiation.
In the present invention, erythrocyte progenitor cells may be obtained from various sources such as peripheral blood, cord blood, or bone marrow. The erythrocyte progenitor cells may be erythroid cells before enucleation. In addition, the erythrocyte progenitor cells may be proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythrocytes, or mixtures thereof.
In one embodiment, the erythrocyte progenitor cells may be CD71 positive (CD71+) cells or glycophorin A positive (GPA+) cells, and may be cells differentiated from hematopoietic stem cells into erythroid cells by treatment with erythropoietin. CD71+ cells or GPA+ cells as erythrocyte progenitor cells may be isolated by various cell isolation methods known in the art, for example, an immunomagnetic-bead isolation method using CD71+ antibodies.
In one embodiment, the erythrocyte progenitor cells may be cells derived from cord blood, bone marrow or peripheral blood.
Erythrocyte progenitor cells differentiate into mature erythrocytes through the erythropoiesis process consisting of the following stages: (a) differentiation from hematopoietic stem cells into proerythroblasts; (b) differentiation from proerythroblasts into basophilic erythroblasts; (c) differentiation from basophilic erythroblasts into polychromatic erythroblasts; (e) differentiation from polychromatic erythroblasts into orthochromatic erythroblasts; and (f) differentiation from orthochromatic erythroblasts to erythrocytes (red blood cells) via reticulocytes.
In the method according to the present invention, the erythrocyte progenitor cells may be cultured by an appropriate method known in the art or a modified method thereof. More specifically, the erythrocyte progenitor cells may be cultured in a stroma-free medium. As the stroma-free medium, any medium capable of growing blood cells may be used as a basic medium, and for example, Iscove's modified dextrose media (IMDM); Stemline II hematopoietic stem cell expansion media (Sigma) specialized for blood cells; X-vivo media (Lonza, M D, USA), or Stemspan media (Stem Cell Technologies) may be used. Reagents added to the media are not very limited, and for example, reagents used in [Baek E J, Kim H S et al. 2009] may be included, but are not limited thereto. In one embodiment of the present invention, the medium may be a serum-free, plasma-free and stroma-free medium including albumin, transferrin, ferric nitrate, insulin, L-glutamine and monothioglycerol, that is, a Stemline II hematopoietic stem cell expansion medium or Iscove's modified dextrose media containing the aforementioned components.
Furthermore, the medium used in the present invention may include vitamin C so as to maintain a stable state against oxidative stress in vitro, may further include an antibiotic such as penicillin, streptomycin or gentamycin, if necessary, and may further include at least one among a stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, a granulocyte macrophage colony stimulating factor, a macrophage colony stimulating factor, a granulocyte colony stimulating factor or erythropoietin.
The method of the present invention includes the step of, during the culture of erythrocyte progenitor cells, converting the cultured cells into an agitation culture type when the diameter of the erythrocyte progenitor cells reaches 10 to 15 μm, for example, 11 to 14 μm, and 12 to 13 μm.
In the above step, the erythroid cells when converted into the agitation culture type may include erythroid cells in the growth and maturation phases.
As in the present invention, when the diameter of erythrocyte progenitor cells reaches a range of 10 to 15 μm, in the case where the culture cells are converted into the agitation culture type, cells with the diameter size as described above have reached the maturation phase, so that in this case, when the agitation culture type is applied, not only cell proliferation occurs well, but also excellent cell viability and an excellent enucleation rate may be exhibited.
In the present invention, erythrocyte progenitor cells may be stationary-cultured in a culture vessel before the diameter of erythrocyte progenitor cells reaches a range of 10 to 15 μm.
In the present invention, the stationary culture refers to culturing in a state of being allowed to remain in a culture vessel without agitating or shaking, is performed under conditions free from hydrodynamic shear stress except when media are exchanged, and is also called 2D culture or planar culture. However, stationary culture in the present invention may also include those performed in bioreactors or agitators. In this case, the “free from hydrodynamic shear stress” means that the hydrodynamic shear stress is substantially an agitation speed of less than 30 rpm or a tip speed of less than 0.018 m/s. For example, it may mean that the hydrodynamic shear stress during the stationary culture is substantially an agitation speed of less than 10 rpm or a tip speed of less than 0.006 m/s.
In the present invention, the tip speed may be determined by the revolutions per minute (rpm) of a bioreactor or agitator and the diameter of a rotating blade, and can be specifically shown in Table 1 according to the following General Formula 1.
Tip speed=Revolutions per minute (rpm)×0.262×Diameter of rotating blade (inch) [General Formula]
Since it has been described in many documents that the tip speed is proportional to the hydrodynamic shear stress (Kim Gail Clarke., Bioprocess Engineering, 9—Bioprocess scale up, 2013, pages 171-188), the description of the hydrodynamic shear stress in the present invention in terms of tip speed and agitation speed (or revolutions per minute) will correspond to the self-obvious level for those skilled in the art. In the present invention, the stationary culture may be performed at a temperature of 20 to 38° C., for example, 30 to 37° C., but is not limited thereto.
A culture vessel that can be used for the stationary culture used in the present invention may include a flask, a T-flask, a disposable cell culture bag, an agitator or a bioreactor, but is not limited thereto.
In the present invention, agitation culture refers to 3D culture (three-dimensional culture), and may be performed under conditions where there is hydrodynamic shear stress due to medium flow. In this case, the “there is a hydrodynamic shear stress” means that the hydrodynamic shear stress is an agitation speed of 10 rpm or more or a tip speed of 0.006 m/s or more. Further, the agitation culture (3D culture) is a method capable of culturing cells at a high cell concentration per standard volume such that cells can be accumulated in one or more layers, and agitation culture may involve culturing cells at high density in order to efficiently utilize the space and medium required for cell culture. The agitation culture may increase the cell density of erythroid cells by smoothly supplying oxygen and nutrients to the cells through agitation of the medium. For example, the cell density may be in a range of 1×104 cells/mL to 5×107 cells/mL, more preferably 1×105 cells/mL to 1×107 cells/mL, and for example, 0.5×106 cells/mL to 5×106 cells/mL. When erythroid cells are cultured at the cell density as described above, the proliferation rate and enucleation rate of erythroid cells may be very high. In addition, the agitation culture may be performed at a temperature condition of 28 to 38° C., for example, 30 to 37° C., depending on the cell maturation period, but is not limited thereto. Furthermore, in the agitation culture, cells may be cultured at an agitation speed of 50 to 700 rpm, for example, 100 to 650 rpm, 200 to 650 rpm, and 300 to 600 rpm, and may be cultured at a tip speed of 0.03 m/s to 0.42 m/s, for example, 0.06 m/s to 0.39 m/s, 0.12 m/s to 0.39 m/s, and 0.18 m/s to 0.36 m/s. In this case, in agitation culture, erythroid cells before enucleation may be agitation-cultured at 35 to 38° C., and when the enucleation process in in vitro culture is imminent, cell death (apoptosis) of erythroid cells increases and cell viability drops sharply, but in vitro culture may be performed at 28 to 33° C. after 15 days of culture because the maturation of erythroid cells may be stabilized by lowering the culture temperature (Kim H O & Baek E J., Tissue engineering part A, 2012; 18 (1-2):117-26.).
In one embodiment, red blood cells produced according to the present invention were found to have a GPA+/CD71−/nuclei-type similar to that of fresh peripheral blood red blood cells (PB RBC). This means that the red blood cells according to the present invention correspond to mature red blood cells. Further, it was confirmed that the red blood cells produced according to the present invention were successfully stored in a refrigerator for 21 days and most of the red blood cells maintained their biconcave red blood cell shape well (Example 3 and
Therefore, the agitation culture according to the present invention may be for obtaining fully mature red blood cells.
A reaction vessel that can be used for the agitation culture used in the present invention may include a shaking flask, a shaking incubator, a fermentation tank, a T-flask, a disposable cell culture bag, a bioreactor, but is not limited thereto.
In one embodiment, the reactor may be a bioreactor.
In one embodiment, the agitation culture may be performed under conditions of a temperature of 30 to 37° C., a dissolved oxygen (DO) of 10 to 50%, and an agitation speed of 250 to 500 rpm or a tip speed of 0.15 to 0.30 m/s.
The reactor for stationary culture and the reactor for agitation culture may be the same or different. For example, when the reactor for stationary culture and the reactor for agitation culture are the same, after the stationary culture is completed in the same reactor, cells can be cultured by an agitation culture method by additionally supplying a medium including necessary nutrients such as cytokines. In addition, when the reactor for stationary culture and the reactor for agitation culture are different, after the stationary culture is completed, a culture may be transferred to the reactor for agitation culture to perform the agitation culture.
In conventional studies to produce red blood cells in vitro, red blood cells have been produced by culturing under sparging-free constant conditions under conditions of a pH of 7.5, a dissolved oxygen (DO) of 50% and 450 rpm (0.27 m/s) in a microbioreactor until hematopoietic stem cells become red blood cells. However, in the case of conventional studies, the step of manually confirming the shape and condition of the cells by an expert during the process of producing red blood cells was essentially performed, and even with this, there was no objective indication of when and under what conditions it should be placed in the incubator, so that it was difficult to perform automated process culture. However, the method according to the present invention divides the in vitro red blood cell production process into maturation phases (growth phase, maturation phase, and enucleation phase) with different cell properties to establish detailed process conditions for each phase, and optimal culture conditions for each subdivided phase were derived. The method for in vitro production of red blood cells according to the present invention enables automated process culture without the need for an expert to confirm the shape of cells each time by specifying the maturation phase of the cells, which exhibits the optimum effect in agitation culture by the cell size.
Furthermore, the present invention provides a blood preparation including red blood cells produced by the method for in vitro production of red blood cells as described above.
In the following examples, it was confirmed that the red blood cells produced in vitro by the method of the present invention not only have an excellent oxygen-carrying capacity similar to that of freshly donated blood, but also have an excellent degree of deformation under pressure compared to general red blood cells, and thus, red blood cells have the same level of function as red blood cells.
Therefore, red blood cells produced according to the present invention may have various therapeutic actions.
In one embodiment, the red blood cells may be used for transfusion. The ability to produce large amounts of cells for transfusion can alleviate the chronic shortage of blood experienced by blood banks and hospitals nationwide. The method of the present invention allows the production of universal cells for transfusion.
A specific aspect of the present invention relates to the expansion of human red blood cells to reach commercial amounts.
Human red blood cells are produced on a large scale, stored as needed, and supplied to hospitals, clinicians, or other health care facilities. Once a patient shows indications such as, for example, ischemia or vascular injury, or requires hematopoietic reconstitution, human red blood cells may be ordered and supplied in a timely manner. Therefore, the present invention relates to a method for generating and expanding human red blood cells to reach commercial scale, a cell preparation including human red blood cells derived from the above method, and a method for providing human red blood cells to hospitals and clinicians (that is, producing, optionally storing and selling).
Additionally, another specific aspect of the present invention relates to a method for producing, storing and distributing red blood cells produced by the method described in the present invention. After the generation and expansion of human red blood cells ex vivo or in vitro, the human red blood cells may be harvested, purified, and optionally stored prior to patient treatment. Therefore, in one embodiment, the present invention provides a method for supplying red blood cells to hospitals, health care centers, and clinicians, whereby red blood cells produced by the method described in the present invention are stored, ordered on demand by hospitals, health care centers or clinicians, and administered to patients in need of red blood cell therapy.
The benefits and features of the present invention, and the methods of achieving the benefits and features will become apparent with reference to embodiments to be described below in detail. However, the present invention is not limited to the exemplary embodiments to be disclosed below, and may be implemented in various other forms, and the present exemplary embodiments are only provided for rendering the disclosure of the present invention complete and for fully representing the scope of the invention to a person with ordinary skill in the technical field to which the present invention pertains, and the present invention will be defined only by the scope of the claims.
The present invention relates to a method for in vitro production of red blood cells. The method for in vitro production of red blood cells, according to the present invention, specifies, by cell size, the maturation stage of cells exhibiting optimal effects in an agitation-type culture to select the time point of an agitation culture, so that even if an expert does not always identify the cell shape, automated processes of culturing are possible, and thus automation in bioreactors is possible in the mass-production of uniform quality and red blood cells.
Hereinafter, the present application will be described in detail through examples. The following examples only illustrate the present application, and the scope of the present application is not limited to the following examples
Since erythroid cells at the immature phase are vulnerable to shearing stress, the artificial erythrocyte production culture using hematopoietic stem cells or hematopoietic precursor cells is performed not by an agitation method but by a weak medium flow method. Examples of the weak medium flow method include a method of initiating stationary culture (2D culture process) on a cell culture plate, and then changing to an agitation culture process when the cell number and cell concentration increase. In this case, the corresponding process may be divided into an immature phase, a growth phase, a maturation phase and an enucleation phase, and may be shown as a detailed process for each phase (
The development of the culture process was performed by establishing a process plan using the design of experiment technique, and the effectiveness of a model was confirmed through statistical analysis of the correlation between process results and process factors, and an optimum value was derived. After switching to a bioreactor, the optimization of process conditions was performed for each step during the continuous culture process.
1) Agitation Culture of Erythroid Progenitor Cells at Immature Phase
Frozen cord blood hematopoietic stem cells (CD34+ cells) were cultured in a Stemline II medium (Sigma Aldrich, St Louis, MO), which is a hematopoietic stem cell proliferation medium, in a 2D flask in a 5% CO2 incubator at 37° C. and a cell inoculation concentration of 1×105 cells/mL, and after the hematopoietic stem cells were cultured, proerythroblasts were put into a continuously stirred-tank bioreactor (Ambr™, Sartorius) on day 7 of culture day, and cultured under agitation at 300 rpm and 600 rpm for 7 days, respectively. In this case, the medium was replaced every two days (50% ratio) while performing agitation culture under conditions of 37° C., a cell inoculation concentration of 0.5×106 cells/mL, a cell solubility of 25%, and a pH of 7.4. For cultured cells, viable cell density (VCD), cell viability and cell diameter were measured (
As a result, there was no significant difference in viable cell density (VCD), cell viability and diameter according to rpm (
Next, in order to investigate whether the culture environment can be improved according to the medium, erythroid progenitor cells were cultured under various cell concentration and medium conditions ([Stemline II+5% plasma]; [Stemline II 80%+DEF-CS (Cellartis DEF-CS 500 Basal Medium, Takara, Kyoto, Japan) 20% (optimized mixed medium (OMM))]; and [Stemline II+DEF-CS™ XENO-FREE GMP Grade Basal Medium (Takara) (OMM (GMP (Good Manufacturing Practice))]. The specific culture conditions are as follows: cells were inoculated at concentrations of 0.5×105 cells/mL and 1.0×106 cells/mL for each medium, and cultured under conditions of 37° C., a pH of 7.4, 300 rpm, and a dissolved oxygen of 25%, and the medium was replaced every 2 days (50% ratio). In this case, OMM means that Stemline : DEF-CS were mixed at a volume ratio of 8:2, and the reagents added to the medium are as follows:
As a result, it could be confirmed that similar to previous experimental results, erythroid progenitor cells at the immature phase could not survive even under various culture conditions (
2) Agitation Culture of Erythroid Cells at Growth and Maturation Phases
In stationary culture (2D culture), erythroid cells cultured under the conditions of [Stemline II+5% FBS] were inoculated into a bioreactor on day 12 of culture (D12) in which basophilic erythroblasts and polychromatic erythroblasts were mixed, and cultured under the [Stemline II+DEF-CS™ XENO-FREE GMP Grade Basal Medium (OMM (GMP))] medium conditions (37° C., a dissolved oxygen of 25%, pH 7.4, a cell inoculation concentration of 1.5×106 cells/mL, 300 rpm, and 50% medium exchange every two days).
As a result, in all the resulting data, erythroid cells matured and grew well, and although there was no significant difference in cell viability or diameter between media, the density (VCD) was higher and the enucleation rate was very high at 94.3% particularly during culture in the OMM medium (Ambr OMM) (
3) Agitation Culture of Erythroid Cells at Proliferation Phase
In the previous experiment 1), erythroid progenitor cells at the immature phase were cultured in stationary culture for 6 days, and then agitation culture was initiated on day 7 (D7) of culture, and in the experiment 2), erythroid cells at the growth and maturation phases were cultured in stationary culture for 11 days, and then agitation culture was initiated on day 12 of culture (D12). Accordingly, in order to specify the starting point of agitation culture of erythroid cells, erythroid progenitor cells were cultured in stationary culture for 9 to 12 days, respectively, and then agitation culture was initiated by inoculation into a bioreactor on day 10 (D10), day 11 (D11), day 12 (D12) and day 13 (D13) of stationary culture. As a result, in the case of erythroid cells, which were inoculated into the bioreactor on day 10 of culture to initiate agitation culture, the cell diameter, which showed an average of 13.14 μm, decreased very rapidly in one day, this was interpreted as erythroid cells at the immature phase quickly died in an agitated environment (
In addition, through the tendency of cell size at each inoculation time to decrease, it was understood that cells inoculated in the bioreactor on days 11 and 12 showed similar cell size results because cell proliferation occurred at this time (
Furthermore,
The conditions for in vitro production of red blood cells presented in the present invention were performed by designing experimental conditions based on the statistics-based design of experiment (DoE), and the present invention derived optimal process conditions by deriving correlations between experimental results and process factors as multivariate statistical functions.
1) Determination of Cell Inoculation Day and Inoculated Cell Concentration During Agitation Culture (Growth of Basophilic Erythroblasts and Polychromatic Erythroblasts)
Erythroid cells at the maturation phase (basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts and erythrocytes) that had reached culture days 12, 13 and 14 in stationary culture were inoculated into a bioreactor, and cultured on an OMM medium. The erythroid cells were cultured under conditions of 37° C., a dissolved oxygen (DO) of 25%, and a pH of 7.4 with 50% medium exchange every two days, and process results were evaluated by enumerating viable cell density (VCD), cell viability, cell diameter and polychromatic red blood cells and orthochromatic erythroblasts. In order to evaluate process results during culture, viable cell density (VCD), cell viability and cell diameter were measured by reading approximately 4,000 to 5,500 cells per sample measurement using Vi-Cell XR (Beckman Coulter, Miami, Florida, USA) for sampled cells.
Cell culture process parameters and DoE conditions are shown in
However, this optimal value is a value compared regardless of the culture period in the bioreactor after inoculation (
Meanwhile, the cell size on inoculation days 12 and 13 of inoculation into the bioreactor was 12.79±0.13 μm, and the cell size on inoculation day 14 was 10.68±0.13 μm, showing a significant difference (
The functional evaluation of red blood cells produced according to the present invention was performed after harvesting cells cultured in a continuously stirred-tank bioreactor (Ambr) on day 21. After only pure red blood cells were filtered through a filter (
As a result, the EI values of peripheral blood of a healthy person and produced red blood cells were 0.31% and 0.29%, respectively, based on the pressure of 3 Pa, showing no difference in deformability, and exhibited better values than red blood cells produced by 2D culture (
Furthermore, in order to verify the oxygen-carrying capacity, p50 values were measured and compared with those of the peripheral blood of a healthy person as a control from oxygen equilibrium curves using a Hemox analyzer (TCS Scientific) (
As a result, it was confirmed that the oxygen-carrying capacity of the red blood cells produced according to the present invention appeared to be similar to that of the peripheral blood control of a healthy person (red blood cells produced according to the present invention p50=24.9; control p50=30.9). The red blood cells produced according to the present invention were successfully stored in a refrigerator for 21 days and most of the red blood cells maintained their biconcave red blood cell shape well (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0105419 | Aug 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/010880 | 8/17/2021 | WO |
