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CN108220241B - Erythrocyte progenitor cell serum-free medium and use method thereof - Google Patents

Erythrocyte progenitor cell serum-free medium and use method thereof Download PDF

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CN108220241B
CN108220241B CN201711458658.7A CN201711458658A CN108220241B CN 108220241 B CN108220241 B CN 108220241B CN 201711458658 A CN201711458658 A CN 201711458658A CN 108220241 B CN108220241 B CN 108220241B
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erythrocyte progenitor
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俞君英
董成友
张颖
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Anhui Zhongsheng Traceability Biotechnology Co ltd
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Abstract

本发明属于培养基领域，具体涉及一种红细胞祖细胞无血清培养基，包括基础培养基以及其他添加成分；所述其他添加成分包括ITS添加剂、GlutaMAX、Lipid Concentrate、L‑抗坏血酸2‑磷酸化半镁盐水合物、无机盐、硫辛酸、氢化可的松、干细胞因子、促红细胞生成素和白细胞介素‑3。本发明的有益效果是：整个培养基体系中的成分明确，品质稳定，选择性地扩增血细胞中的红细胞祖细胞，使得红细胞祖细胞在保持其原来干性特征的基础上实现有效表达特异性标志CD71+/CD235a+/–，且CD71+/CD235a+/–比例占约50％～100％，本发明培养基适用于临床应用和科研研究中。

Figure 201711458658

The invention belongs to the field of culture medium, and in particular relates to a serum-free medium for erythrocyte progenitor cells, comprising a basal medium and other supplementary components; the other supplementary components include ITS additives, GlutaMAX, Lipid Concentrate, L-ascorbic acid 2-phosphorylated half Magnesium salt hydrate, inorganic salts, lipoic acid, hydrocortisone, stem cell factor, erythropoietin and interleukin-3. The beneficial effects of the invention are: the composition of the whole culture medium system is clear, the quality is stable, the erythrocyte progenitor cells in the blood cells are selectively amplified, so that the erythrocyte progenitor cells can achieve effective expression specificity on the basis of maintaining their original stemness characteristics Marking CD71+/CD235a+/–, and the ratio of CD71+/CD235a+/– accounts for about 50% to 100%, the culture medium of the present invention is suitable for clinical application and scientific research.

Figure 201711458658

Description

Erythrocyte progenitor cell serum-free medium and use method thereof
Technical Field
The invention belongs to the field of culture media, and particularly relates to a serum-free culture medium for erythrocyte progenitors and a using method thereof.
Background
Induced pluripotent stem cells (ipscs) are an important source of stem cells for replacing embryonic stem cells for the treatment of diseases, the study of embryonic developmental stages and the detection of disease mechanisms. This is particularly important for the study of human embryonic development, disease mechanisms and treatments, since ethical problems involving the use of human embryos are avoided.
Human ipscs (hipscs) were initially generated by overexpressing several transcription factors in dermal fibroblasts (Takahashi et al, 2007; Yu et al, 2007; Park et al, 2008). Like human embryonic stem cells (hescs), hipscs are capable of differentiating into functional cell lineages and thus have great potential for disease models, drug screening, and future therapeutic medicine (Kondo et al, 2013; Park et al, 2008).
Takahashi k1.,et al.,Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell.2007 Nov 30；131(5):861-872.
Yu,J.,et al.,Induced pluripotent stem cell lines derived from human somatic cells.Science.2007；318:1917-1920.
Park IH.,et al.,Reprogramming of human somaic cells to pluripotency with defined factors.Nature.2008；451:141-146.
Recent studies have used human urine samples for the derivation of epithelial-like cells that are capable of reconstituting (reprogrammad) pluripotency (Zhou et al, 2011). This method provides an unlimited source of cells that can be conveniently and non-invasively collected. Nevertheless, this method requires the collection of large volumes of urine samples, leading to problems of logistical difficulties in downstream storage and transport, susceptibility to contamination by microorganisms, etc. (Xue et al, 2013).
Zhou T.,er al.,Generation of induced pluripotent stem cells from urine.J Am Soc Nephrol.2011 Jul；22(7):1221-8.
Xue Y.,et al.,Generating a non-integrating human induced pluripotent Stem cell bank from urine-derived cells.PLoS One.2013 Aug 5；8(8):e70573.
In contrast, peripheral blood T cells are readily obtained from donors and reconstituted with high efficiency (Lohn et al, 2010; Seki et al, 2010; Staerk et al, 2010). However, T cell-derived hipscs carry an existing v (d) J rearrangement on the T Cell Receptor (TCR) gene, and it is not yet clear whether these hipscs can differentiate normally or whether such cells demonstrate a trend towards T cell lymphoma (Serwold et al, 2007).
Loh YH.,et al.,Reprogramming of T cells from human peripheral blood.Cell Stem Cell.2010 Jul 2；7(1):15-9.
Seki T.,et al.,Generation of induced pluripotent stem cells from human terminallydifferentiated circulating T cells.Cell Stem Cell.2010 Jul 2；7(1):11-4.
Staerk J.,et al.,Reprogramming of human peripheral blood cells to induced Pluripotent stem cells.Cell Stem Cell.2010 Jul 2:7(1):20-4.
Serwold T.,er al.,Early TCR expression and aberrant T cell development in mice with endogenous prerearranged T cell receptor genes.J Immunol.2007 Jul 15；179(2):928-38.
Recent studies have reported the reconstitution of non-T cell populations in peripheral blood, (Chou et al, 2011; Mack et al, 2011; Okita et al, 2013), but do not involve the reconstitution of erythroid progenitors, because under normal conditions the number of erythroid progenitors is very small, about 500-1000/ml in peripheral blood, the number in cord blood is about 3.5 times higher, human erythroid progenitors cannot be expanded to some extent, and there are limitations in maintaining dryness and number.
Chou BK.,et al.,Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigeneic and gene expression signatures.Cell Res.2011 Mar；21(3):518-29.
Mack AA.,et al.,Generation of induced pluripotent stem cells from CD34+cells across blood drawn from multiple donors with non-integrating episomal vectors.PLoS One.2011；6(11)；e27956.
Okita K.,er al.,An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.Stem Cells.2013 Mar；31(3)；458-66.
Disclosure of Invention
In order to solve the above problems, the present invention provides a serum-free culture medium for erythrocyte progenitors, which can selectively and efficiently amplify erythrocyte progenitors in a blood sample.
The invention provides the following technical scheme:
a serum-free culture medium for erythrocyte progenitor cells comprises a basal culture medium and other additives;
the other additive components comprise ITS additive, GlutaMAX, Lipid Concentrate, magnesium sesquimagnesium L-ascorbate 2-phosphate hydrate, inorganic salt, lipoic acid, hydrocortisone, stem cell factor, erythropoietin and interleukin-3.
Preferably, the Basal Medium is selected from any one of Dulbecco's Modified Eagle Medium, Minimum Essential Medium, basic Medium Eagle, Glasgow's Minimum Essential Medium, α Minimum Essential Medium, Iscove's Modified Dulbecco's Medium, F-10, F-12, RPMI1640, and M199.
Preferably, the basic Medium is Iscove's Modified Dulbecco's Medium.
Preferably, the inorganic salt is selected from one or two of ferrous sulfate and ferric nitrate ferrous sulfate.
Preferably, each liter of the amplification medium comprises 1-100ml of ITS additive, 0.1-50ml of GlutaMAX, 0.1-50ml of Lipid Concentrate, 100-300 mu mol of L-ascorbic acid 2-magnesium sesquiphosphate magnesium salt hydrate, 1-5 mu mol of ferrous sulfate, 0.1-2 mu mol of ferric nitrate, 0.1-2 mu mol of lipoic acid, 0.1-2 mu mol of hydrocortisone, 10-200 mu g of dry cell factor, 1-50 mu g of erythropoietin and 31-10 mu g of interleukin.
Preferably, each liter of the amplification medium comprises 2.5-40ml of ITS additive, 0.5-20ml of GlutaMAX, 0.5-10ml of Lipid Concentrate, 150-280 mu mol of L-ascorbic acid 2-magnesium phosphate sesquimagnesium salt hydrate, 2-5 mu mol of ferrous sulfate, 0.1-2 mu mol of ferric nitrate, 0.1-1 mu mol of lipoic acid, 0.3-1.5 mu mol of hydrocortisone, 70-200 mu g of dry cell factor, 5-30 mu g of erythropoietin and 33-10 mu g of interleukin.
The invention also provides a serum-free culture medium for the erythrocyte progenitor cells, which is suitable for the preparation and application of the erythrocyte progenitor cells in human erythrocytes.
The invention also provides a use method of the erythrocyte progenitor cell serum-free medium, which comprises the following steps:
s1, collecting a blood sample;
s2, separating different blood components in the blood sample collected in the S1 by using a gradient centrifugation method to obtain mononuclear cells;
s3, carrying out selective amplification culture on the mononuclear cells obtained in the S2 in an erythrocyte progenitor cell serum-free culture medium to obtain erythrocyte progenitor cells containing a CD71+/CD235a + specific marker.
Preferably, the blood sample is stored at 3 ℃ to 5 ℃ or on ice for less than or equal to 48 hours prior to performing the blood component separation.
Preferably, in the step S3, the selective amplification culture is performed for 6 to 16 days.
Preferably, in the step S3, the erythrocyte progenitors containing the specific marker CD71+/CD235a +/-account for 50-100% of the total number of the erythrocyte progenitors after the expansion culture.
The invention has the beneficial effects that:
exogenous animal-derived components are not introduced into the whole culture medium system, erythrocyte progenitor cells are selectively and efficiently amplified for blood samples with convenient sources, and the effective expression of a specific marker CD71+/CD235a +/-is realized on the basis of keeping the original dry characteristic.
Drawings
FIG. 1 is a graph of the morphology of cells cultured in formula A medium;
FIG. 2A shows the results of a 10-day CD71 expression experiment in formula A medium; wherein the light gray line is the antibody negative control and the black line is the percentage expression of CD71 antibody in the erythrocyte progenitor cells;
FIG. 2B shows the results of a 10-day CD235a expression experiment in formula A medium cultured cells, where the light gray line indicates the antibody negative control and the black line indicates the percentage of CD235a antibody expression in erythrocyte progenitors;
FIG. 3 is a graph of the total cell number obtained after counting the expansion of erythrocyte progenitors after 1 day, 8 days and 16 days of culture in formula A medium and control medium.
Detailed Description
The invention provides a serum-free culture medium for erythrocyte progenitors, which comprises a basic culture medium and other additive components; other additional ingredients include ITS additive, GlutaMAX, Lipid Concentrate, magnesium sesquimagnesium L-ascorbate 2-phosphate hydrate, inorganic salts, lipoic acid, hydrocortisone, stem cell factor, erythropoietin, and interleukin-3.
The erythrocyte progenitor cell serum-free medium can specifically induce and expand erythrocyte progenitor cells from mononuclear cells, and the differentiation and expansion of other cells are inhibited, so that the erythrocyte progenitor cells specifically expressed by CD71+/CD235a +/-can be effectively and massively expanded on the basis of keeping the original dryness characteristics, and the problems of the source and the quantity of the precursor cells reprogrammed by hipscs can be solved.
Blood samples collected by the present invention include human peripheral blood, umbilical cord blood, bone marrow blood, and the like, and the volume of the blood sample used is minute, for example, from about 10 μ l to about 10ml, or any volume falling within this range and greater than 10 ml. Once collected, the blood sample may be stored on ice, or may be refrigerated at a temperature of about 3 ℃ to about 5 ℃ or about 4 ℃. Thus, the sample may be stored on ice for no more than about 8 hours, no more than about 12 hours, no more than about 16 hours, no more than about 20 hours, no more than about 24 hours, no more than about 28 hours, no more than about 32 hours, no more than about 36 hours, no more than about 40 hours, no more than about 44 hours, or no more than about 48 hours. If the sample is to be stored for more than 48 hours, the sample is frozen using liquid nitrogen and stored for future use.
Processing the collected blood sample by using a gradient centrifugation method, separating different blood components, and discarding components such as red blood cells to obtain mononuclear cells; the mononuclear cells include neutrophils, eosinophils, basophils, lymphocytes, erythrocyte progenitors, and the like. Culturing the obtained mononuclear cells with the erythrocyte progenitor cell serum-free medium of the invention is beneficial to the survival of the contained erythrocyte progenitor cells and effectively expands the obtained sufficient cell number to allow the effective induction of pluripotency. The length of the amplification period may vary and amplification may be over a period of no more than 16 days, for example from about 6 to about 14 days. The ratio of CD71+/CD235a +/present in the expanded cell population may be at least half to increase the efficiency of pluripotency induction, e.g., the expanded cell population may contain about 50% or more of CD71+/CD235a +/-, about 75% or more of CD71+/CD235a +/-, about 80% or more of CD71+/CD235a +/-, about 85% or more of CD71+/CD235a +/-, about 90% or more of CD71+/CD235a +/-, about 95% or more of CD71+/CD235a +/-, upon completion of the expansion period. The remaining blood from the sample can be used for other types of analysis, for example, a blood sample remaining after gradient centrifugation can be used for mycoplasma and/or virus screening, and analysis of the blood provides a comprehensive assessment of donor health.
The present invention will be described in detail with reference to the following examples.
The reagents used in the following examples are as follows:
IMDM (basal medium, Thermo fisher), ITS additive (Sigma), GlutaMAX (Thermo fisher), Lipid Concentrate (Thermo fisher), magnesium sesqui-magnesium L-Ascorbate (Mg Ascorbate), ferrous sulfate (FeSO)4) Iron nitrate (Fe (NO)3)3) Lipoic Acid (Lipoic Acid), hydrocortisone (Tocri), stem cell factor SCF (PeproTech), erythropoietin EPO (PeproTech), interleukin-3 IL-3(PeproTech), Stemspan (Stemcell).
Example 1: erythrocyte progenitor cell serum-free medium
IMDM medium (serum-free) was used as the basal medium to which was added a composition selected from the formula in any one of columns A, B, C, D, E in table 2:
TABLE 1 formulation of erythrocyte progenitor cell serum-free medium in different proportions
Figure GDA0002830645160000061
Figure GDA0002830645160000071
Example 2 Primary isolation and culture of erythrocyte progenitors
Collecting 10ml adult peripheral blood, transferring into lymphocyte separation tube, centrifuging, and collectingWashing mononuclear cell layer with DPBS twice, sampling, counting, and taking 0.5 × 106Inoculating cells/ml in 6-well plate, dividing into six groups, wherein 5 groups are experimental groups, 1 group is control group, 5 groups are experimental groups, and are added with erythrocyte progenitor cell serum-free culture medium at a ratio shown in Table 1, and control group is added with Stemspan culture medium at 2 ml/well, placing the 6 groups of cells at 37 deg.C and 5% CO2Culturing in an incubator. The 5 experimental groups are respectively supplemented with 2ml of fresh erythrocyte progenitor cell serum-free culture medium on the 4 th day of amplification, the control group is correspondingly added with Stemspan culture medium, the 5 experimental groups are respectively supplemented with 2ml of fresh erythrocyte progenitor cell serum-free culture medium after 2ml of culture medium is discarded on the 8 th day and 12 th day of amplification, and the control group is correspondingly added with Stemspan culture medium.
Example 3 identification of erythrocyte progenitors
As shown in FIG. 1, the change process of the cell morphology of the erythrocyte progenitor cells is observed by a microscope at 1 day, 8 days and 16 days, and the change process is photographed and stored, and the scale bar of the microscope is 200 μm.
As shown in FIG. 2, the expression of surface molecules CD71 and CD235a was detected by flow cytometry at day 10 of erythrocyte progenitor cell culture, and the positive rate and the average fluorescence intensity index were respectively detected.
As shown in FIG. 3, the total cell number obtained after the expansion of the erythrocyte progenitor cells was counted after 1 day, 8 days and 16 days of culture, EP-GM was the erythrocyte progenitor cell culture medium using formulation A in Table 1, and Stemspan was the control group.
As a result: observing the expansion of the separated blood mononuclear cells in an erythrocyte progenitor cell culture medium system by an inverted microscope, wherein the number of erythrocyte progenitor cell colonies is the largest when the blood mononuclear cells are cultured for 7 days, then gradually reducing, indicating that the blood mononuclear cells are in an erythrocyte progenitor cell proliferation stage in 0-7 days, then entering a differentiation stage, and gradually increasing the expression of erythrocyte progenitor cell specific markers CD71+ and CD235+ and increasing the cell number as the culture time is prolonged.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1.一种红细胞祖细胞无血清培养基，其特征在于，由基础培养基以及其他添加成分组成；1. an erythrocyte progenitor cell serum-free medium, is characterized in that, is made up of basal medium and other additive components; 所述基础培养基为Iscove's Modified Dulbecco's Medium；Described basal medium is Iscove's Modified Dulbecco's Medium; 所述红细胞祖细胞无血清培养基为ITS添加剂10ml、GlutaMAX 10ml、LipidConcentrate 1ml、L-抗坏血酸2-磷酸酯镁倍半镁盐水合物250μmol、硫酸亚铁3μmol、硝酸铁0.2μmol、硫辛酸1μmol、氢化可的松1μmol、干细胞因子SCF 100μg、促红细胞生成素EPO20μg和白细胞介素-3 5μg，使用基础培养基补足至一升。The serum-free medium for erythrocyte progenitor cells is ITS additive 10ml, GlutaMAX 10ml, LipidConcentrate 1ml, L-ascorbic acid 2-phosphate magnesium sesquimagnesium salt hydrate 250μmol, ferrous sulfate 3μmol, ferric nitrate 0.2μmol, lipoic acid 1μmol, Hydrocortisone 1 μmol, stem cell factor SCF 100 μg, erythropoietin EPO 20 μg and interleukin-3 5 μg, make up to one liter with basal medium. 2.一种如权利要求1所述的红细胞祖细胞无血清培养基在人红细胞祖细胞制备中的应用。2. The application of the erythrocyte progenitor serum-free medium as claimed in claim 1 in the preparation of human erythrocyte progenitor cells. 3.一种如权利要求1所述的红细胞祖细胞无血清培养基的使用方法，其特征在于，所述方法包括：3. A method of using the serum-free medium for erythrocyte progenitor cells as claimed in claim 1, wherein the method comprises: S1、收集血液样本；S1. Collect blood samples; S2、使用梯度离心方法将S1中收集的血液样本中不同的血液成分分离，获得单个核细胞；S2. Use gradient centrifugation to separate different blood components in the blood sample collected in S1 to obtain mononuclear cells; S3、将S2中获得的单个核细胞在红细胞祖细胞无血清培养基中进行选择性扩增培养，获得含有CD71+/CD235a+特异性标志的红细胞祖细胞。S3. The mononuclear cells obtained in S2 are selectively expanded and cultured in erythrocyte progenitor serum-free medium to obtain erythrocyte progenitor cells containing CD71 + /CD235a + specific markers. 4.根据权利要求3所述的一种红细胞祖细胞无血清培养基的使用方法，其特征在于，所述血液样本在进行血液成分分离之前，在3℃至5℃冰上存储小于或等于48小时。4. The method for using a serum-free medium for erythrocyte progenitor cells according to claim 3, wherein the blood sample is stored on ice at 3°C to 5°C and less than or equal to 48°C prior to the separation of blood components. Hour. 5.根据权利要求3或4所述的一种红细胞祖细胞无血清培养基的使用方法，其特征在于，所述S3步骤中，进行选择性扩增培养的持续时间为6至16天。5. The method for using a serum-free medium for erythrocyte progenitor cells according to claim 3 or 4, characterized in that, in the step S3, the duration of the selective expansion culture is 6 to 16 days. 6.根据权利要求3或4或所述的一种红细胞祖细胞无血清培养基的使用方法，其特征在于，所述S3步骤中，含有CD71+/CD235a+/-特异性标志的红细胞祖细胞占扩增培养后红细胞祖细胞总数的50～100％。6. according to claim 3 or 4 or the use method of the described a kind of erythrocyte progenitor serum-free medium, it is characterized in that, in described S3 step, the erythrocyte progenitor cell containing CD71 + /CD235a +/- specific mark It accounts for 50-100% of the total number of erythrocyte progenitor cells after expansion and culture.
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