Denucleation stage culture medium and system for inducing erythroblast terminal differentiation

Abstract

The present disclosure provides a denucleation stage culture medium and system for inducing erythroblast terminal differentiation. The culture medium comprises a basic culture medium, a lipid, holo-transferrin and erythropoietin, wherein the culture medium does not contain serum or plasma. Preferably, the culture medium is composed of HPLM, 10 μg / mL low-density lipoprotein or 2.5 μM cholesterol or 5% lipid supplement, 500 μg / mL holo-transferrin and 3 U / mL erythropoietin. The present disclosure also provides a method for using the culture medium to prepare mature red blood cells in vitro.

Description

Enucleation stage culture medium and culture system for inducing terminal differentiation of erythroblasts Technical Field

[0001] The present disclosure relates to the field of cell biology, and more specifically, to a culture medium and culture system for inducing terminal differentiation of erythroblasts during the enucleation stage, which does not require the addition of human plasma or serum and has clear culture medium components. Background Art

[0002] Red blood cells (RBCs) are the most numerous type of cell in human blood and are the main medium for oxygen transport. Red blood cell transfusion plays an important role in surgery, anemia, large-scale trauma and cancer treatment. However, the global blood supply is in serious shortage and unevenly distributed. There are about 100 million units of blood products in the world (each unit of blood product contains about 2×10 12 However, with the breakthrough of research related to in vivo erythropoiesis and the development of in vitro red blood cell culture and preparation technology, the problem of blood shortage is expected to be solved.

[0003] The in vitro preparation of mature human red blood cells (RBCs) consists of two main stages: erythroblast expansion and terminal differentiation, maturation, and denucleation. The erythroblast expansion stage involves the massive expansion of RBCs from various sources, such as umbilical cord blood, bone marrow, and peripheral blood, in a culture system. The terminal differentiation and maturation stage primarily involves the gradual maturation and denucleation of RBCs into reticulocytes under terminal differentiation-inducing culture conditions. The two stages serve different purposes, and therefore require different supplements to achieve different effects.

[0004] In the past, the in vitro preparation of red blood cells required the addition of human plasma or serum. Only when human plasma or serum is added to the terminal differentiation culture system can erythroblasts undergo the normal gradual differentiation, maturation and denucleation process. However, the essential properties of human plasma or serum in the terminal maturation process pose a huge challenge to the in vitro production of mature red blood cells. First, in China, human plasma or serum products are strictly prohibited from commercialization, which has hindered the realization of large-scale, low-cost in vitro red blood cell production; second, as biological products, plasma or serum has unclear composition and large individual differences, resulting in unstable in vitro red blood cell production efficiency; third, the pathogens that may be carried by plasma or serum pose potential safety risks to the in vitro production and clinical application of mature red blood cells. Therefore, establishing a red blood cell preparation system with clear composition and no need to add plasma or serum, and optimizing the production process, are key issues that need to be urgently addressed.

[0005] Summary of the Invention

[0006] One aspect of the present disclosure provides an enucleation stage culture medium for inducing terminal differentiation of erythroblasts, comprising a basal culture medium, lipids, holo-transferrin and erythropoietin (EPO), wherein the enucleation stage culture medium does not contain serum or plasma.

[0007] According to an embodiment of the present disclosure, the basal culture medium is selected from HPLM, IMDM, SFEM, DMEM or RPMI 1640.

[0008] According to an embodiment of the present disclosure, the lipid is selected from low-density lipoprotein, cholesterol or lipid supplements; wherein, when the lipid is selected from cholesterol or lipid supplements, the enucleation stage culture medium further includes methyl-β-cyclodextrin.

[0009] According to an embodiment of the present disclosure, the above-mentioned lipid is selected from low-density lipoprotein with a concentration of 2 to 100 μg / mL, preferably 4 to 20 μg / mL, and more preferably 10 μg / mL, cholesterol with a concentration of 0.5 to 20 μM, preferably 2.5 μM, or a lipid supplement with a concentration (volume fraction) of 1% to 20%, preferably 5%; wherein, when the above-mentioned lipid is selected from cholesterol or a lipid supplement, the above-mentioned enucleation stage culture medium further includes methyl-β-cyclodextrin with a concentration of 0.01 to 1 mg / mL, preferably 0.1 mg / mL.

[0010] According to an embodiment of the present disclosure, the concentration of the above-mentioned holo-transferrin is 50-1000 μg / mL, preferably 500 μg / mL.

[0011] According to an embodiment of the present disclosure, the concentration of the erythropoietin is 1 to 10 U / mL, preferably 3 U / mL.

[0012] Another aspect of the present disclosure provides an enucleation stage culture system for inducing terminal differentiation of erythroblasts, comprising: a first-stage culture medium, including a basal culture medium, lipids, holo-transferrin and erythropoietin, wherein the first-stage culture medium does not contain serum or plasma; a second-stage culture medium, including the above-mentioned basal culture medium, the above-mentioned lipids and the above-mentioned holo-transferrin, wherein the second-stage culture medium does not contain serum or plasma; and a third-stage culture medium, including the above-mentioned basal culture medium, wherein the third-stage culture medium does not contain serum or plasma; wherein the above-mentioned first-stage culture medium, the above-mentioned second-stage culture medium and the above-mentioned third-stage culture medium are suitable for use at different stages of terminal differentiation culture of erythroblasts.

[0013] According to an embodiment of the present disclosure, the basal culture medium is selected from HPLM, IMDM, SFEM, DMEM or RPMI 1640.

[0014] According to an embodiment of the present disclosure, the lipid is selected from low-density lipoprotein, cholesterol or lipid supplements; wherein, when the lipid is selected from cholesterol or lipid supplements, the enucleation stage culture medium further includes methyl-β-cyclodextrin.

[0015] According to an embodiment of the present disclosure, the above-mentioned lipid is selected from low-density lipoprotein with a concentration of 2 to 100 μg / mL, preferably 10 μg / mL, cholesterol with a concentration of 0.5 to 20 μM, preferably 2.5 μM, or a lipid supplement with a concentration of 1% to 20%, preferably 5%.

[0016] According to an embodiment of the present disclosure, the concentration of the above-mentioned holo-transferrin is 50-1000 μg / mL, preferably 500 μg / mL.

[0017] According to an embodiment of the present disclosure, the concentration of the erythropoietin is 1 to 10 U / mL, preferably 3 U / mL.

[0018] Yet another aspect of the present disclosure provides a method for preparing mature red blood cells in vitro using the enucleation stage culture system as described above, comprising: performing a first-stage culture on erythroblasts using a first-stage culture medium; replacing the medium of the cells obtained from the first-stage culture using a second-stage culture medium, and performing a second-stage culture; replacing the medium of the cells obtained from the second-stage culture using a third-stage culture medium, and performing a third-stage culture; and separating mature red blood cells from the cell culture fluid obtained from the third-stage culture.

[0019] According to an embodiment of the present disclosure, the conditions for the above-mentioned first stage culture include: culture at 37°C, 5% CO2 for 2 to 5 days, for example, 3 to 4 days, for example, 3 days; the conditions for the above-mentioned second stage culture include: culture at 37°C, 5% CO2 for 2 to 5 days, for example, 3 to 4 days, for example, 3 days; the conditions for the above-mentioned third stage culture include: culture at 37°C, 5% CO2 for 1 to 3 days, for example, 2 days.

[0020] According to the embodiment of the present disclosure, the number of the erythroblasts added to each 1 mL of the first stage culture medium is 0.1×10 6 ~0.3×10 6 , preferably 0.2×10 6 .

[0021] According to an embodiment of the present disclosure, separating mature red blood cells from the cell culture medium cultured in the third stage includes:

[0022] The cell culture fluid obtained from the third stage of culture is filtered through a leukocyte filter, and the cells in the filtrate are the mature red blood cells.

[0023] According to an embodiment of the present disclosure, the erythroblasts are obtained by culturing original or genetically modified human peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, bone marrow cells or hematopoietic stem / progenitor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 shows the erythrocyte yield results of Example 1-1 of the present disclosure based on inducing terminal differentiation of erythroblasts under different basal culture medium conditions.

[0025] FIG2 shows the erythrocyte yield results of inducing terminal differentiation of erythroblasts by adding different concentrations of serum in Examples 1-2 of the present disclosure.

[0026] FIG3 shows the erythrocyte yield results of inducing terminal differentiation of erythroblasts by adding different concentrations of low-density lipoprotein in Examples 1-2 of the present disclosure.

[0027] FIG4 shows the erythrocyte yield results of Examples 1-3 of the present disclosure when different concentrations of cholesterol were added to induce terminal differentiation of erythroblasts.

[0028] FIG5 shows the erythrocyte yield results of Examples 1-4 of the present disclosure in which different concentrations of lipid supplements were added to induce terminal differentiation of erythroblasts.

[0029] FIG6 shows the erythrocyte yield results of Examples 1-5 of the present disclosure when different concentrations of holo-transferrin were added to induce terminal differentiation of erythroblasts.

[0030] FIG7 shows the erythrocyte yield results of the induced erythroblast terminal differentiation in Example 2 of the present disclosure before and after optimization of the culture system for the enucleation stage and in Comparative Example 1.

[0031] FIG8 is a flow chart of preparing mature red blood cells in vitro in Example 3 of the present disclosure.

[0032] FIG9 is a flow cytometry test result of preparing mature red blood cells in vitro in Example 3 of the present disclosure.

[0033] FIG10 is the Wright-Giemsa staining result of mature red blood cells prepared in vitro in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0035] The present disclosure provides a culture medium and culture system for inducing terminal differentiation of erythroblasts during the enucleation phase. During the implementation of this disclosure, it was discovered that the addition of lipids can replace plasma or serum, and combined with holotransferrin and erythropoietin, can stably and efficiently promote erythrocyte differentiation, maturation, and enucleation to form mature erythrocytes. This has led to the establishment of a well-defined erythrocyte culture medium and culture system that does not require the addition of plasma or serum, and optimizes the in vitro method for preparing mature erythrocytes.

[0036] definition:

[0037] The term "erythroblasts" used in this article is also called "erythroblasts", "erythroblasts" or "erythroid progenitor cells". They are nucleated red blood cells that can be cultured from original or genetically modified human peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, bone marrow cells and hematopoietic stem cells.

[0038] The term "reticulocyte" used in this article refers to mature red blood cells obtained by denucleation after erythroblasts differentiate and mature to the point where they no longer divide. Reticulocytes contain a small amount of ribonucleic acid (RNA), and further maturation can produce fully mature red blood cells in which RNA disappears.

[0039] Specifically, according to some embodiments of the present disclosure, a denucleation stage culture medium for inducing terminal differentiation of erythroblasts is provided, comprising a basal culture medium, lipids, holo-transferrin and erythropoietin, wherein the denucleation stage culture medium does not contain serum or plasma.

[0040] According to the embodiments of the present disclosure, in the presence of the above-mentioned basal culture medium, holotransferrin and erythropoietin, if plasma or serum is replaced with lipids, a similar effect of promoting erythroblast differentiation and denucleation to form mature red blood cells can be achieved.

[0041] According to an embodiment of the present disclosure, the above-mentioned basal culture medium is selected from HPLM, IMDM, SFEM, DMEM or RPMI 1640. Preferably, the above-mentioned basal culture medium is HPLM.

[0042] According to an embodiment of the present disclosure, the lipid is selected from low-density lipoprotein (LDL) at a concentration of 2 to 100 μg / mL, preferably 4 to 20 μg / mL, and more preferably 10 μg / mL, cholesterol at a concentration of 0.5 to 20 μM, preferably 1 to 2.5 μM, and more preferably 2.5 μM, or a lipid supplement at a concentration (volume fraction) of 1% to 20%, preferably 5% to 10%, and more preferably 5%; wherein when the lipid is selected from cholesterol or a lipid supplement, the enucleation stage culture medium further includes methyl-β-cyclodextrin at a concentration of 0.01 to 1 mg / mL, preferably 0.1 mg / mL, in the presence of methyl-β-cyclodextrin, so that the lipid can be effectively taken up by erythroblasts and thus exert its effect. Too high or too low a concentration of lipid will have an adverse effect on the yield of mature red blood cells.

[0043] According to the embodiments of the present disclosure, the concentration of holotransferrin is 50-1000 μg / mL, preferably 500 μg / mL. Homotransferrin is a key growth factor that promotes erythroblast differentiation, maturation, and denucleation. Too high or too low a concentration will adversely affect the yield of mature erythrocytes.

[0044] According to the embodiments of the present disclosure, the concentration of erythropoietin is 1-10 U / mL, preferably 3 U / mL. Erythropoietin is a key growth factor that promotes erythroblast differentiation, maturation, and denucleation. Too high or too low a concentration will adversely affect the yield of mature red blood cells.

[0045] According to some embodiments of the present disclosure, there is also provided a method for preparing mature red blood cells in vitro using the enucleation stage culture medium as described above, comprising:

[0046] Step A) culturing erythroblasts using the above enucleation stage culture medium, and changing the medium during the culture process; Step B) separating mature erythrocytes from the cell culture medium obtained by culture.

[0047] According to an embodiment of the present disclosure, in step A), the number of erythroblasts added to each 1 mL of the above enucleation stage culture medium is 0.1×10 6 ~0.3×10 6 , preferably 0.2×10 6 .

[0048] According to an embodiment of the present disclosure, step A) specifically includes: step A1) using the above-mentioned enucleation stage medium to culture erythroblasts for 2 to 5 days, preferably 3 to 4 days, more preferably 3 days; step A2) using the above-mentioned enucleation stage medium to replace the medium for culturing the cells obtained by culturing in step A1) for 2 to 5 days, preferably 3 to 4 days, more preferably 3 days; step A3) using the above-mentioned enucleation stage medium to replace the medium for culturing the cells obtained by culturing in step A2) for 1-3 days, preferably 2 days.

[0049] According to an embodiment of the present disclosure, further, the culture conditions of steps A1) to A3) include: culture at 37° C. and 5% CO 2 .

[0050] According to an embodiment of the present disclosure, step B) specifically includes: filtering the cell culture fluid obtained by the above-mentioned culture with a leukocyte filter, and the cells in the obtained filtrate are mature red blood cells.

[0051] Based on the aforementioned enucleation stage culture medium, the role of each component in inducing terminal erythroblast differentiation was also studied. By optimizing the timing and concentration of each component addition during the terminal differentiation stage, costs could be further reduced without affecting red blood cell yield. This resulted in the establishment of an enucleation stage culture system for inducing terminal erythroblast differentiation.

[0052] According to some embodiments of the present disclosure, a denucleation stage culture system for inducing terminal differentiation of erythroblasts is provided, comprising: a first-stage culture medium, including a basal culture medium, lipids, holotransferrin and erythropoietin, wherein the first-stage culture medium does not contain serum or plasma; a second-stage culture medium, including the above-mentioned basal culture medium, the above-mentioned lipids and holotransferrin, wherein the second-stage culture medium does not contain serum or plasma; and a third-stage culture medium, including the above-mentioned basal culture medium, wherein the third-stage culture medium does not contain serum or plasma; wherein the above-mentioned first-stage culture medium, the above-mentioned second-stage culture medium and the above-mentioned third-stage culture medium are suitable for use in different stages of terminal differentiation culture of erythroblasts.

[0053] According to the embodiments of the present disclosure, the first-stage culture medium is the above-mentioned enucleation stage culture medium, the second-stage culture medium is based on the first-stage culture medium without adding erythropoietin, and the third-stage culture medium is based on the first-stage culture medium without adding lipids, holotransferrin and erythropoietin. Experiments have shown that this optimized culture system has no obvious adverse effect on the yield of mature red blood cells and has greater economic benefits.

[0054] According to the embodiments of the present disclosure, the first stage culture medium, the second stage culture medium and the third stage culture medium constitute a terminal differentiation culture system, wherein the types and concentrations of the components are the same as those of the above-mentioned enucleation stage culture medium, and therefore are not described in detail here.

[0055] According to some embodiments of the present disclosure, a method for preparing mature red blood cells in vitro using the enucleation stage culture system as described above is also provided, comprising: step a) performing a first-stage culture of the erythroblasts using a first-stage culture medium; step b) replacing the medium of the cells obtained from the first-stage culture using a second-stage culture medium, and performing a second-stage culture; step c) replacing the medium of the cells obtained from the second-stage culture using a third-stage culture medium, and performing a third-stage culture; and step d) separating mature red blood cells from the cell culture fluid obtained from the third-stage culture.

[0056] According to the embodiment of the present disclosure, the number of erythroblasts added to each 1 mL of the first stage culture medium is 0.1×10 6 ~0.3×10 6 , preferably 0.2×10 6 .

[0057] According to the embodiments of the present disclosure, the duration of the first stage culture is 2 to 5 days, preferably 3 to 4 days, more preferably 3 days; the duration of the second stage culture is 2 to 5 days, preferably 3 to 4 days, more preferably 3 days; the duration of the third stage culture is 1-3 days, preferably 2 days.

[0058] According to an embodiment of the present disclosure, the culture conditions of steps a) to c) may include: culturing at 37° C. and 5% CO 2 . The first stage culture medium, the second stage culture medium, and the third stage culture medium may be used in equal amounts.

[0059] According to the embodiments of the present disclosure, by optimizing the culture method in the enucleation stage, the final yield of red blood cells can be improved at a lower cost, with good production and economic benefits.

[0060] The technical solutions of the present disclosure are further described below through specific examples in combination with the accompanying drawings. It should be noted that the following specific examples are only used as examples, and the scope of protection of the present disclosure is not limited thereto. The drugs or reagents used in the following examples are all commercially available or homemade by known preparation methods. The methods used in the following examples, such as Wright-Giemsa staining, are all well-known methods in the art and can be carried out by referring to the descriptions in textbooks or relevant literature, and will not be described in detail.

[0061] Preparation Example Isolation and Expansion of Erythroblasts

[0062] Erythroblasts can be obtained by culturing original or genetically modified human peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, bone marrow cells, and hematopoietic stem / progenitor cells. In this embodiment, erythroblasts are derived from human peripheral blood mononuclear cells, which are inoculated into a directed differentiation induction culture system for induced expansion.

[0063] The specific culture system is as follows, including: StemSpan TM SFEM (STEMCELL Technologies, #09650), GlutaMAX TM (Gibco, #35050079), interleukin-3 (10 ng / mL; STEMCELL Technologies, #78194.1), human recombinant stem cell factor (SCF; 100 ng / mL; STEMCELL Technologies, #78062.2), dexamethasone (DEX; 1 mmol / L; Sigma-Aldrich, #D2915), erythropoietin (EPO; 3 U / mL; R&D Systems, #287-TC). The medium was changed every three days. Erythroblasts were obtained on day 8-10 of culture for subsequent experiments.

[0064] Example 1 Preparation of reticulocytes under different denucleation stage culture medium conditions

[0065] Example 1-1 Selection of different basal culture media

[0066] Complete culture medium was prepared by adding 10% plasma or 10% serum, 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) to three basal culture media: IMDM (Gibco, #21056023), SFEM (STEMCELL Technologies, #09650), and HPLM (Gibco, #A4899101). 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0067] On the 3rd and 6th day of cell culture, the medium was changed, the cells were collected, centrifuged at 300g for 5 minutes, the supernatant was discarded, and an equal amount of the above complete medium was added. The cells were cultured at 37°C and 5% CO2. After culturing for 2 days, the cells were collected and counted in an automatic cell counter to obtain the cell concentration. The cell count was multiplied by the corresponding volume to obtain the final cell count (the total number of enucleated cells and non-enucleated cells). Take 0.2×10 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a + DRAQ5 - The proportion of cells to the total number of cells).

[0068] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. As shown in Figure 1, after multiple verifications, when the same number of cells (0.2×10 6 The reticulocyte yield was the highest when HPLM was used as the basal medium for 8 days.

[0069] Example 1-2 Selecting low-density lipoprotein as a substitute for serum or plasma

[0070] The first stage culture medium was prepared by adding different concentrations (2-100 μg / mL) of low-density lipoprotein (LDL) (YEASEN, #20613ES05) or different concentrations (0-10%) of serum, 500 μg / mL holo-transferrin (R&D Systems, #2914-HT) and 3 U / mL EPO (R&D Systems, #287-TC) to HPLM (Gibco, #A4899101) culture medium. 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0071] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 minutes, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium was supplemented with LDL (YEASEN, #20613ES05) or serum at the same concentration as in the first-stage medium, and 500 μg / mL holo-transferrin (R&D Systems, #2914-HT).

[0072] On the 6th day of culture, the medium was changed and the cells were harvested and centrifuged at 300 g for 5 min. The supernatant was discarded and an equal amount of HPLM (Gibco, #A4899101) was added as the third stage medium. The cells were cultured at 37°C and 5% CO2. After 2 days of culture, the cells were harvested and counted using an automatic cell counter to obtain the cell concentration. The final cell count (total number of enucleated and non-enucleated cells) was obtained by multiplying the cell count by the corresponding volume. 0.2×10 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a + DRAQ5 - The proportion of cells to the total number of cells).

[0073] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. After multiple verifications, as shown in Figure 2, when the same number of cells (0.2×10 6 The cells were cultured for 8 days under the conditions of 1% human serum and 2% human erythroblasts. The reticulocyte yield was the highest under the conditions of 2% human serum. As shown in Figure 3, the reticulocyte yield was the highest under the conditions of 10 μg / mL low-density lipoprotein, which could achieve the same induction differentiation and maturation effect as the 2% human serum conditions.

[0074] Example 1-3: Choosing Cholesterol as a Substitute for Serum or Plasma

[0075] HPLM (Gibco, #A4899101) medium was added with different concentrations (0.5-20 μM) of cholesterol (MCE, #HY-N0322), 0.1 mg / mL methyl-β-cyclodextrin (MCE, #HY-101461), 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) to prepare the first stage medium. 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0076] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 min, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium was supplemented with the same concentrations of cholesterol (MCE, #HY-N0322) as in the first-stage medium, 0.1 mg / mL methyl-β-cyclodextrin (MCE, #HY-101461), and 500 μg / mL holo-transferrin (R&D Systems, #2914-HT). On day 6 of culture, the medium was changed again, the cells were harvested, centrifuged at 300 g for 5 min, the supernatant discarded, and an equal amount of HPLM (Gibco, #A4899101) was added as the third-stage medium. The cells were cultured at 37°C in 5% CO2. After culturing for 2 days, cells were collected and counted in an automatic cell counter to obtain the cell concentration, which was multiplied by the corresponding volume to obtain the final cell number (the total number of enucleated cells and non-enucleated cells). 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a + DRAQ5 - The proportion of cells to the total number of cells).

[0077] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. After multiple verifications, as shown in Figure 4, when the same number of cells (0.2×10 6 The reticulocyte yield was highest under 1-2.5 μM cholesterol conditions, which could achieve the same induction differentiation and maturation effect as that under 2% human serum conditions.

[0078] Examples 1-4 Selecting lipid supplements as a substitute for serum or plasma

[0079] To HPLM (Gibco, #A4899101) medium, lipid supplement (Gibco, #11905031) at varying concentrations (1% to 20%), 0.1 mg / mL methyl-β-cyclodextrin (MCE, #HY-101461), 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) were added to prepare the first stage medium. To each mL of the first stage medium, 0.2 × 10 6 The cells were cultured at 37°C in 5% CO2.

[0080] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 minutes, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium was supplemented with the same concentration of lipid supplement (Gibco, #11905031) as in the first-stage medium, 0.1 mg / mL methyl-β-cyclodextrin (MCE, #HY-101461), and 500 μg / mL holo-transferrin (R&D Systems, #2914-HT).

[0081] On the 6th day of culture, the medium was changed and the cells were harvested and centrifuged at 300 g for 5 min. The supernatant was discarded and an equal amount of HPLM (Gibco, #A4899101) was added as the third stage medium. The cells were cultured at 37°C and 5% CO2. After 2 days of culture, the cells were harvested and counted using an automatic cell counter to obtain the cell concentration. The final cell count (total number of enucleated and non-enucleated cells) was obtained by multiplying the cell count by the corresponding volume. 0.2×10 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a +DRAQ5 - The proportion of cells to the total number of cells).

[0082] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. After multiple verifications, as shown in Figure 5, when the same number of cells (0.2×10 6 The reticulocyte yield was the highest under the condition of 5% lipid supplement, which could achieve the same induction differentiation and maturation effect as that under the condition of 2% human serum.

[0083] Example 1-5 Selecting different concentrations of whole transferrin

[0084] The first stage culture medium was prepared by adding 10 μg / mL low-density lipoprotein (LDL) (YEASEN, #20613ES05), different concentrations (50 μg / mL or 500 μg / mL) of holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) to HPLM (Gibco, #A4899101) culture medium. 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0085] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 minutes, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium supplemented with 10 μg / mL LDL (YEASEN, #20613ES05) and various concentrations of holo-transferrin (R&D Systems, #2914-HT).

[0086] On the 6th day of culture, the medium was changed and the cells were harvested and centrifuged at 300 g for 5 min. The supernatant was discarded and an equal amount of HPLM (Gibco, #A4899101) was added as the third stage medium. The cells were cultured at 37°C and 5% CO2. After 2 days of culture, the cells were harvested and counted using an automatic cell counter to obtain the cell concentration. The final cell count (total number of enucleated and non-enucleated cells) was obtained by multiplying the cell count by the corresponding volume. 0.2×10 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a + DRAQ5 - The proportion of cells to the total number of cells).

[0087] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. After multiple verifications, as shown in Figure 6, when the same number of cells (0.2×10 6 The reticulocyte yield was highest when 500 μg / mL holo-transferrin was added from day 0 to day 6.

[0088] Example 2 Preparation of reticulocytes under different denucleation stage culture system conditions

[0089] Example 2-1 Culture medium system for the initial enucleation stage

[0090] Terminal differentiation medium was prepared by adding 10 μg / mL low-density lipoprotein, 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) to HPLM (Gibco, #A4899101) medium.

[0091] Add 0.2×10 6 Erythroblasts were cultured at 37°C, 5% CO2. On days 3 and 6 of culture, the medium was changed, the cells were harvested, centrifuged at 300g for 5 minutes, the supernatant discarded, and an equal amount of the above-mentioned terminal differentiation medium was added. The cells were cultured at 37°C, 5% CO2. After 2 days of culture, the cells were harvested.

[0092] Example 2-2 Optimization of the culture medium system for the enucleation stage

[0093] To HPLM (Gibco, #A4899101) medium, 10 μg / mL low-density lipoprotein (LDL) (YEASEN, #20613ES05), 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) were added to prepare the first stage medium. 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0094] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 minutes, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium supplemented with 10 μg / mL LDL (YEASEN, #20613ES05) and 500 μg / mL holo-transferrin (R&D Systems, #2914-HT).

[0095] On the sixth day of culture, the medium was changed and the cells were harvested and centrifuged at 300 g for 5 min. The supernatant was discarded and an equal amount of HPLM (Gibco, #A4899101) was added as the third stage medium. The cells were cultured at 37°C in 5% CO2. The cells were harvested after 2 days of culture.

[0096] Comparative Example 1

[0097] Terminal differentiation medium was prepared by adding 10% human plasma, 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), 3 U / mL EPO (R&D Systems, #287-TC), 10 mg / ml insulin, and 4 U / ml heparin to IMDM (Gibco, #21056023) medium.

[0098] Add 0.2×10 6 Erythroblasts were cultured at 37°C, 5% CO2. On days 3 and 6 of culture, the medium was changed, the cells were harvested, centrifuged at 300g for 5 minutes, the supernatant discarded, and an equal amount of the above-mentioned terminal differentiation medium was added. The cells were cultured at 37°C, 5% CO2. After 2 days of culture, the cells were harvested.

[0099] The cells collected from Example 2-1, Example 2-2 and Comparative Example 1 were counted in an automatic cell counter to obtain the cell concentration, which was multiplied by the corresponding volume to obtain the final cell number (the total number of enucleated cells and non-enucleated cells). 6 The cells were centrifuged and the precipitate was collected and resuspended in 20 μL PBS. 1 μL CD235a and 0.1 μL DRAQ5 were added and incubated at 4°C in the dark for 30 min. The denucleation rate (CD235a + DRAQ5 - The proportion of cells to the total number of cells).

[0100] The denucleation rate was analyzed by cell counting and flow cytometry, and the final red blood cell yield was obtained by statistical analysis according to the final red blood cell yield = number of cells × denucleation rate. After multiple verifications, as shown in Figure 7, when the same number of cells (0.2×10 6 Compared with the culture medium system in the initial enucleation stage, the optimized enucleation stage culture medium system simplifies the system composition, but can still ensure normal and stable red blood cell production. Compared with the culture system of comparative example 1, the final red blood cell production of the culture system of the present disclosure is increased by 3-4 times.

[0101] Example 3 Preparation of reticulocytes

[0102] This example uses the process shown in Figure 8 to prepare reticulocytes in vitro:

[0103] To HPLM (Gibco, #A4899101) medium, 10 μg / mL low-density lipoprotein (LDL) (YEASEN, #20613ES05), 500 μg / mL holo-transferrin (R&D Systems, #2914-HT), and 3 U / mL EPO (R&D Systems, #287-TC) were added to prepare the first stage medium. 0.2×10 6 The cells were cultured at 37°C in 5% CO2.

[0104] After 3 days of cell culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 minutes, the supernatant discarded, and an equal amount of second-stage medium was added. The cells were cultured at 37°C in 5% CO2. The second-stage medium was prepared as follows: HPLM (Gibco, #A4899101) medium supplemented with 10 μg / mL LDL (YEASEN, #20613ES05) and 500 μg / mL holo-transferrin (R&D Systems, #2914-HT).

[0105] On the sixth day of culture, the medium was changed, the cells were harvested, centrifuged at 300 g for 5 min, the supernatant discarded, and an equal amount of HPLM (Gibco, #A4899101) was added as the third stage medium. The cells were cultured at 37°C, 5% CO2. After 2 days of culture, the cells were harvested, centrifuged at 300 g for 5 min, the supernatant discarded, and the cells were resuspended in PBS and filtered through a leukocyte filter.

[0106] Flow cytometry was used to detect the red blood cells in the filtrate obtained in this example. As shown in Figure 9, the collected cells did not contain nuclei (DRAQ5 negative) and expressed CD235a. The above results are consistent with the results reported (Heshusius S, Heideveld E, Burger P, Thiel-ValkhofM, Sellink E, Varga E, et al. Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells. Blood Adv. 2019; 3(21): 3337-50. and Liu S, Wu M, Lancelot M, Deng J, Gao Y, Roback JD, et al. BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells. Mol Ther. 2021; 29(5): 1918-32.). The red blood cells prepared in vitro were DRAQ5 negative and CD235a positive, which is consistent with the specific protein characteristics of normal reticulocytes.

[0107] The collected red blood cells were examined using Wright-Giemsa staining. As shown in Figure 10, the collected cells lacked nuclei. These results are consistent with those published by Heshusius S, Liu S, et al. Wright-Giemsa staining of in vitro prepared red blood cells revealed a lack of nuclei and a pale pink cytoplasm, consistent with normal reticulocyte morphology.

[0108] According to the above embodiments of the present disclosure, the enucleation stage culture medium and culture system for inducing terminal differentiation of erythroblasts disclosed herein have at least one or part of the following beneficial effects:

[0109] The lipids added to the culture medium in the enucleation stage of the present invention can replace plasma or serum, and together with holotransferrin and erythropoietin, can stably and efficiently promote the differentiation, maturation, and enucleation of erythroblasts to form mature red blood cells, thus solving the problem of the source of plasma or serum. Moreover, the culture medium and culture system in the enucleation stage of the present invention have clear components and meet GMP-grade production standards.

[0110] The present invention optimizes the timing and concentration of adding each component to the culture medium during the enucleation stage. As the terminal differentiation culture of erythroblasts progresses, the cost is reduced by controlling the addition of erythropoietin, lipids and holotransferrin at different stages, without affecting the stable and efficient production of mature red blood cells.

[0111] The specific embodiments of the present disclosure described above do not limit the scope of protection of the present disclosure. Any other corresponding changes and modifications made based on the technical concept of the present disclosure should be included in the scope of protection of the claims of the present disclosure.

Claims

1. A denucleation stage culture medium for inducing terminal differentiation of erythroblasts, comprising a basal culture medium, lipids, holo-transferrin and erythropoietin, wherein the denucleation stage culture medium does not contain serum or plasma.

2. The enucleation stage medium according to claim 1, wherein The basal culture medium is selected from HPLM, IMDM, SFEM, DMEM or RPMI 1640.

3. The enucleation stage medium according to claim 1 or 2, wherein The lipid is selected from a low-density lipoprotein with a concentration of 2 to 100 μg / mL, a cholesterol with a concentration of 0.5 to 20 μM, or a lipid supplement with a concentration of 1% to 20%, wherein when the lipid is selected from cholesterol or a lipid supplement, the enucleation stage culture medium further includes methyl-β-cyclodextrin with a concentration of 0.01 to 1 mg / mL.

4. The enucleation stage medium according to claim 1 or 2, wherein The lipid is selected from low-density lipoprotein with a concentration of 10 μg / mL, cholesterol with a concentration of 2.5 μM, or a lipid supplement with a concentration of 5%; Wherein, when the lipid is selected from cholesterol or a lipid supplement, the enucleation stage culture medium further comprises methyl-β-cyclodextrin at a concentration of 0.1 mg / mL.

5. The enucleation stage culture medium according to claim 1 or 2, wherein: The concentration of the holo-transferrin is 50-1000 μg / mL; The concentration of the erythropoietin is 1-10 U / mL.

6. A culture system for inducing terminal differentiation of erythroblasts at the enucleation stage, comprising: a first phase culture medium comprising a basal medium, lipids, holo-transferrin, and erythropoietin, wherein the first phase culture medium does not contain serum or plasma; a second phase culture medium comprising the basal medium, the lipid, and the holo-transferrin, wherein the second phase culture medium does not contain serum or plasma; as well as a third stage medium, comprising the basal medium, wherein the third stage medium does not contain serum or plasma; The first-stage culture medium, the second-stage culture medium and the third-stage culture medium are suitable for use in different stages of the erythroblast terminal differentiation culture.

7. The denucleation stage culture system according to claim 6, wherein: The basal culture medium is selected from HPLM, IMDM, SFEM, DMEM or RPMI 1640.

8. The denucleation stage culture system according to claim 6 or 7, wherein: The lipid is selected from low-density lipoprotein with a concentration of 2 to 100 μg / mL, cholesterol with a concentration of 0.5 to 20 μM, or a lipid supplement with a concentration of 1% to 20%; Wherein, when the lipid is selected from cholesterol or a lipid supplement, the enucleation stage culture medium further comprises methyl-β-cyclodextrin at a concentration of 0.01 to 1 mg / mL.

9. The denucleation stage culture system according to claim 6 or 7, wherein: The lipid is selected from low-density lipoprotein with a concentration of 10 μg / mL, cholesterol with a concentration of 2.5 μM, or a lipid supplement with a concentration of 5%; Wherein, when the lipid is selected from cholesterol or a lipid supplement, the enucleation stage culture medium further comprises methyl-β-cyclodextrin at a concentration of 0.1 mg / mL.

10. The denucleation stage culture system according to claim 6 or 7, wherein: The concentration of the holo-transferrin is 50-1000 μg / mL; The concentration of the erythropoietin is 1-10 U / mL.

11. A method for preparing mature erythrocytes in vitro using the enucleation stage culture system according to any one of claims 6 to 10, comprising: Perform the first stage culture of erythroblasts using the first stage medium; Using the second stage culture medium to replace the medium of the cells obtained by the first stage culture, and then carry out the second stage culture; Using the third stage culture medium to replace the medium of the cells obtained by the second stage culture, and then carry out the third stage culture; The mature red blood cells are separated from the cell culture fluid obtained from the third stage culture.

12. The method according to claim 11, wherein: The conditions of the first stage of culture include: culturing at 37°C, 5% CO2 for 2 to 5 days; The conditions of the second stage culture include: culturing at 37°C, 5% CO2 for 2 to 5 days; The conditions of the third stage culture include: culture at 37° C. and 5% CO 2 for 1 to 3 days.

13. The method according to claim 11, wherein: The number of erythroblasts added to each 1 mL of the first stage culture medium was 0.1×10 6 ~0.3×10 6 .

14. The method according to claim 11, wherein: The method of separating mature red blood cells from the cell culture fluid of the third stage culture comprises: The cell culture fluid obtained from the third stage of culture is filtered through a leukocyte filter, and the cells in the obtained filtrate are the mature red blood cells.

15. The method according to claim 11, wherein: The erythroblasts are obtained by culturing original or genetically modified human peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, bone marrow cells or hematopoietic stem / progenitor cells.

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