WO2019196128A1 - 扩增造血干细胞的培养体系、方法及其用途 - Google Patents

扩增造血干细胞的培养体系、方法及其用途 Download PDF

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WO2019196128A1
WO2019196128A1 PCT/CN2018/083216 CN2018083216W WO2019196128A1 WO 2019196128 A1 WO2019196128 A1 WO 2019196128A1 CN 2018083216 W CN2018083216 W CN 2018083216W WO 2019196128 A1 WO2019196128 A1 WO 2019196128A1
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hematopoietic stem
stem cells
cell culture
culture medium
sodium butyrate
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郭潇
孙忠杰
陈立功
薛庆磊
王晓芳
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诺未科技（北京）有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

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  • the present invention relates to the field of cell culture technology, and in particular to a culture system, method and use thereof for expanding hematopoietic stem cells.
  • Hematopoietic stem cells are by far the only type of stem cell widely used in clinical treatment. Hematopoietic stem cell transplantation technology can treat various blood diseases such as leukemia and lymphoma, and even have effects on metabolic diseases, congenital immunodeficiency, diabetes and other diseases. According to statistics, the annual number of cases receiving hematopoietic stem cell transplantation in the world exceeds 40,000 cases, most of which are derived from the donor's bone marrow and mobilized peripheral blood stem cells; despite the great success of the technology, 70% of patients cannot get the right fit due to the strict matching requirements of HLA matching Donor.
  • Umbilical cord blood hematopoietic stem cells have relatively low requirements for HLA matching and low immunogenicity. Together with their convenient access and abundant sources, they become a major source of hematopoietic stem cell transplantation donors. Since the first umbilical cord blood stem cell transplantation in the 1980s successfully cured Faconi boys, the case of cord blood hematopoietic stem cell transplantation has increased year by year. According to statistics, there are more than 30,000 cases of cord blood hematopoietic stem cell transplantation in the world, and it is still increasing.
  • the bottleneck of cord blood hematopoietic stem cell transplantation technology is that its cell content is small, and the amount of hematopoietic stem cells and progenitor cells contained in a cord blood is not enough to quickly restore the immune system of adult patients, resulting in an increase in the chance of death from opportunistic infection.
  • the current interim strategy is a double cord blood transplant, in which one patient receives two cord blood transplants after clearing the marrow, but this increases the difficulty of matching the donor's HLA matching. Therefore, it is urgent to expand cord blood hematopoietic stem cells.
  • the method is to obtain a sufficient amount of hematopoietic stem cells for transplantation.
  • DLL1, DSL1, etc. can be used to appropriately amplify hematopoietic stem cells by activating Notch signals.
  • PTN secreted by bone marrow endothelial stromal cells can also slightly amplify hematopoietic stem cells.
  • hematopoietic stem cells are under hypoxic conditions, and oxygen stress produced by in vitro culture can impair the self-renewal and transplantation function of hematopoietic stem cells by increasing ROS levels; it has been found that the addition of antioxidants and inhibition of mTOR can offset these damages.
  • SR1-expanded hematopoietic stem cells have the ability to rebuild the patient's immune system, but still do not get rid of the dependence on double cord blood transplantation.
  • methods for regulating a single signaling pathway may be detrimental to the overall regulation of the physiological state of HSC.
  • the present invention proposes a culture system for amplifying hematopoietic stem cells, which can significantly increase the total amount of cells obtained by in vitro expansion of fresh blood-like hematopoietic stem cells.
  • a culture system for amplifying hematopoietic stem cells comprising a cell culture medium containing sodium butyrate having a molecular formula of C4H7NaO2 and a molecular weight of 110.0869.
  • the concentration of sodium butyrate is 50 to 200 ⁇ mol/L.
  • the concentration of sodium butyrate is 100 ⁇ mol/L.
  • the cell culture medium further contains a cytokine, which is at least one of TPO, SCF, and FLT3L.
  • the cell culture medium is StemSpan SFEM II medium containing sodium butyrate, TPO, SCF and FLT3L.
  • the concentration of the TPO in the cell culture medium is 30-70 ng/mL; the SCF is 80-120 ng/mL in the cell culture medium; the concentration of the FLT3L in the cell culture medium is 90-110 ng. /mL.
  • a method for in vitro expansion of hematopoietic stem cells wherein fresh blood-like hematopoietic stem cells are obtained as sample cells, and the hematopoietic stem cells are obtained by in vitro expansion culture using the above-described culture system for expanding hematopoietic stem cells, usually, Fresh blood samples of hematopoietic stem cells can be derived from bone marrow, peripheral blood, and cord blood.
  • the method for in vitro expansion of hematopoietic stem cells specifically includes the following steps:
  • step S2 adding the sodium butyrate concentrated solution obtained in step S1 to StemSpan SFEM II minimal medium to obtain a cell culture medium;
  • the purity of the sodium butyrate in the S1 is ⁇ 98.5%, and the concentration of the sodium butyrate concentrated solution is 200 mol/L.
  • the concentrated solution is directly used and diluted according to the sample volume. The concentrated solution must be stored at -20 ° C.
  • step S3 further includes the following steps:
  • S31 separating fresh blood samples into mononuclear cells by using a lymphocyte separation solution, and obtaining CD34+ cells, ie, hematopoietic stem cells, by MACS, and then storing the hematopoietic stem cells in liquid nitrogen;
  • the culture system for amplifying hematopoietic stem cells is for use in preparing hematopoietic stem cells using fresh blood-like hematopoietic stem cells as sample cells.
  • the culture conditions in the step S3 are: culture at 37 ° C under a condition of a CO 2 concentration of 5%.
  • the culture time in the step S3 is 7-10 days.
  • the expanded hematopoietic stem cells contain a higher proportion of CD34+CD90+ cell populations and have better CFU colony forming ability, indicating that these hematopoietic stem cells are more primitive and have stronger differentiation potential for reconstituting the blood system, which can be more effective. Support clinical treatment needs;
  • Figure 1 is a graph showing the results of statistical analysis of the total number of cells and the total number of CD34+ cells on day 7 of each sample of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 2 is a graph showing the results of total number of CD34+CD45RA- and CD34+CD90+ cells on day 7 of umbilical cord blood hematopoietic stem cells in each concentration of sodium butyrate;
  • Figure 3 is a graph showing the expression of CD34 surface antigen on day 7 of a representative sample of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 4 is a graph showing the expression of CD34 and CD45RA surface antigens on the 7th day of representative samples of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 5 is a graph showing the expression of CD34 and CD90 surface antigens on the 7th day of representative samples of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 6 is a representative map of colony formation of each lineage under an inverted microscope, wherein a picture is CFU-E; b picture is CFU-G; c picture is CFU-M; d picture is CFU-GM; e picture is CFU-GEMM;
  • Fig. 7 is a graph showing the number of colony forming units of each lineage of cord blood hematopoietic stem cell samples on the 7th day of each sodium butyrate culture.
  • StemSpan SFEM II is a serum-free medium supplied by StemCell Technologies under the trade number 09655;
  • RhSCF human stem cell factor
  • Recombinant human FMS-like tyrosine kinase 3 ligand manufactured by Stemimmune LLC, under the accession number HHM-FT-1000;
  • PBMC Peripheral blood mononuclear cell
  • MACS magnetic bead sorting
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffer
  • MethoCult TM GF H4435 a semi-solid medium
  • CFU-E full name Conoly Forming Unit of Erythrocyte Chinese name is red blood cell colony forming unit
  • CFU-G full name Conoly Forming Unit of Granulocyte Chinese name is granulocyte colony forming unit
  • CFU-GM full name Conoly Forming Unit of Granulocyte-Macrophage Chinese name is granulocyte-macrophage colony forming unit
  • CFU-GEMM full name Conoly Forming Unit of granulocyte, erythrocyte, macrophage/monocyte, megakaryocyte, mixed colony, its Chinese name is granulocyte, red blood cell, macrophage/monocyte, megakaryocyte colony forming unit;
  • StemSpan SFEM II medium is available from StemCell Technologies, Inc.
  • rhSCF, rhTPO, rhFLT3L is available from Stemimmune LLC; although the invention is preferred for use in humans, it can also be used in laboratory animals such as mice; human hematopoietic stem cells can be sourced
  • umbilical cord blood hematopoietic stem cells are taken as an example, wherein umbilical cord blood is collected from healthy maternal pregnant infants, and hepatitis B, hepatitis C, syphilis, AIDS, and hepatitis B, hepatitis C, syphilis, AIDS, Cytomegalovirus, TORCH detection, mycoplasma, chlamydia, G-6PD and thalassemia are negative.
  • Human umbilical cord blood hematopoietic stem cells express several membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation antigen CD34, leukocyte differentiation anti
  • the centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, and the second layer is the ring-shaped milky white mononuclear cell layer (PBMC).
  • the third layer is a transparent separation liquid layer, and the fourth layer is an red blood cell layer;
  • PBMC ring-shaped milky white mononuclear cell layer
  • Each umbilical cord blood PBMC was resuspended in a mixture of 50 ul of human CD34+ magnetic beads and 50 ul of FcR blocker reagent and 150 ul of 0.5% BSA, and incubated at 4 ° C for 30 min;
  • the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 80 ng/ml and the FLT3 to FLT3 is added by using StemSpan SFEM II serum-free medium.
  • the concentration was 90 ng/ml, the concentration of TPO to TPO was 30 ng/ml; the cell seeding density in the 24-well plate was 1 ⁇ 10 4 /well, sodium butyrate was added 50 ⁇ M, and the cells were cultured at 37 ° C in a 5% CO 2 incubator.
  • the state of cell culture 500 ⁇ l of the cell culture medium obtained in the step 2 is added every 2 days, and a large number of hematopoietic stem cells can be obtained in 7 to 10 days, and the amplification factor is about 4 to 20 times.
  • the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 100 ng/s using StemSpan SFEM II serum-free medium.
  • Ml add FLT3 to FLT3 at a concentration of 100 ng/ml, add TPO to TPO at a concentration of 50 ng/ml; in a 24-well plate, the cell seeding density is 1 ⁇ 10 4 /well, sodium butyrate is added at 100 ⁇ M, and placed at 37 ° C, 5% Culture in a CO2 incubator.
  • the difference from the first embodiment is that the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 120 ng/s using StemSpan SFEM II serum-free medium.
  • Ml add FLT3 to FLT3 at a concentration of 110 ng/ml, add TPO to TPO at a concentration of 70 ng/ml; in a 24-well plate, the cell seeding density is 1 ⁇ 10 4 /well, sodium butyrate is added at 200 ⁇ M, at 37 ° C, 5% Culture in a CO2 incubator.
  • Example 4 is a blank control group, and the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 80 ng/ml by using StemSpan SFEM II serum-free medium.
  • Table 1 Statistical Table of Number of Conditional Cells in Each Group on Day 7 of CB CD34+ Cell Culture
  • Flow cytometry analysis was performed on different concentrations of sodium butyrate-cultured CD34+ cells on day 0 and day 7, respectively.
  • a FACS Verse flow Detector from BD
  • 20 ⁇ l of the cell suspension was taken, and FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA were added. After vortexing, the tubes were incubated for 15 min at room temperature, added with appropriate amount of PBS, centrifuged at 1600 rpm for 5 min at room temperature, the supernatant was discarded, 200 ⁇ l of PBS was added, and then analyzed by machine.
  • Colony forming unit analysis was performed on different concentrations of sodium butyrate-cultured CD34+ cells on day 0 and day 7, respectively. Using MethoCult TM GF H4435 semi-solid medium, medium 1 ml/well was added to the six-well plate, CD34+ cells were seeded at a density of 1000 cells/well, and placed in a 37 ° C 5% CO 2 incubator for 14 days, and the number of colonies of each lineage was calculated. And take a photo.

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Abstract

一种扩增造血干细胞的培养体系、方法及其用途，包括细胞培养基，所述细胞培养基含有丁酸钠，获取新鲜血样造血干细胞为样本细胞，采用含有丁酸钠的细胞培养基进行体外扩增培养，得到造血干细胞。有益效果：1、通过添加丁酸钠提高了脐带血造血干细胞体外扩增所得的细胞总量，为临床试验提供了前期技术基础；2、扩增后的造血干细胞含有更高比例的CD34+CD90+细胞群，且具有更优异的CFU集落形成能力，表明这些造血干细胞更加原始，具有更强的重建血液系统的分化潜能，可以有效地支持临床治疗需要；3、使得造血干细胞体外扩增更简便安全和高效。

Description

扩增造血干细胞的培养体系、方法及其用途 技术领域
本发明涉及细胞培养技术领域，具体来说，涉及扩增造血干细胞的培养体系、方法及其用途。
背景技术
造血干细胞是迄今为止唯一广泛应用于临床治疗的干细胞类型。造血干细胞移植技术能够治疗白血病、淋巴瘤等多种血液类疾病，甚至能够对代谢性疾病、先天性免疫缺陷、糖尿病等疾病产生疗效；据统计，世界范围内每年接受造血干细胞移植治疗的案例超过40,000例，其中多数造血干细胞的供体来源于捐献者的骨髓和动员的外周血干细胞；尽管该技术取得了巨大的成功，但是由于HLA配型的严格配对要求，70％的患者不能获得合适的供体。
脐带血造血干细胞对于HLA配型的要求相对较低，且免疫原性低，再加上其获取方便、来源丰富，成为造血干细胞移植供体的一大来源。自20世纪80年代首例脐带血造血干细胞移植手术成功治愈Faconi男童患者后，脐带血造血干细胞移植案例逐年增加。据统计，世界范围内脐带血造血干细胞移植案例已超过30,000例，且还在不断增加。目前，脐带血造血干细胞移植技术的瓶颈在于其细胞含量少，一份脐带血中所含的造血干细胞及祖细胞数量不足以快速恢复成人患者的免疫系统，造成机会性感染致死率的增高。目前暂行的策略是双份脐带血移植，即一位患者清髓后先后接受两份脐带血的移植，但这增加了供体的HLA配型匹配难度，因此，亟需扩增脐带血造血干细胞的方法，以获得足量的可供移植的造血干细胞。
人们对于脐带血造血干细胞的体外扩增进行了大量尝试，但都没有取得理想效果。早期人们利用血液中的细胞因子来培养造血干细胞，结果导致细胞分化，而移植功能减弱。后来，人们发现骨髓造血干细胞微环境中的Wnt信号分子、Notch配体、视黄酸拮抗因子等能够有效扩增CD34+造血干/祖细胞。利用CHIR99021或者BIO激活Wnt信号通路维持体外培养的 造血干细胞的移植能力；而在造血干细胞的培养体系中添加DLL1，DSL1等，能够通过激活Notch信号而适度扩增造血干细胞。另有研究发现，骨髓内皮基质细胞分泌的PTN也能够轻微扩增造血干细胞。生理状态下造血干细胞处在低氧条件下，而体外培养产生的氧胁迫会通过增高ROS水平损害造血干细胞的自我更新和移植功能；人们发现，抗氧化剂的添加以及mTOR的抑制能够抵消这些损害。然而，上述技术并没能够显著扩增脐带血造血干细胞。偶然的发现，铜离子螯合剂TEPA，SIRT抑制剂Nicotinamide能够显著提高造血干细胞移植水平，且在临床实验中显示初步疗效，但扩增后的细胞体内存活时间不够长，且分化谱系不够完整。近几年的高通量筛选化学小分子发现一类氮杂环化合物SR1和吲哚类似物UM171能够更有效的扩增具备长期移植能力的造血干细胞。临床实验表明，SR1扩增的造血干细胞具备重建患者免疫系统的能力，但其依然没有摆脱对双份脐带血移植的依赖。总的来说，囿于人们对造血干细胞的有限认识，针对单一信号通路进行调节的方法可能不利于整体调节HSC的生理状态。
针对相关技术中的问题，目前尚未提出有效的解决方案。
发明内容
针对相关技术中的上述技术问题，本发明提出扩增造血干细胞的培养体系，能够显著提高了新鲜血样造血干细胞体外扩增所得的细胞总量。
为实现上述技术目的，本发明的技术方案是这样实现的：
扩增造血干细胞的培养体系，包括细胞培养基，所述细胞培养基含有丁酸钠，分子式为C4H7NaO2，分子量为110.0869。
进一步地，所述细胞培养基中，丁酸钠的浓度为50～200μmol/L。
进一步地，所述细胞培养基中，丁酸钠的浓度为100μmol/L。
进一步地，所述细胞培养基还含有细胞因子，所述细胞因子为TPO、SCF、FLT3L中的至少。
进一步地，所述细胞培养基为含有丁酸钠、TPO、SCF和FLT3L的StemSpan SFEM II培养基。
进一步地，所述TPO在细胞培养基中的浓度为30-70ng/mL；所述SCF在细胞培养基中的为80-120ng/mL；所述FLT3L在细胞培养基中的浓度为 90-110ng/mL。
本发明的另一方面，提供了造血干细胞的体外扩增方法，获取新鲜血样造血干细胞为样本细胞，采用上述所述的扩增造血干细胞的培养体系进行体外扩增培养，得到造血干细胞，通常，造血干细胞的新鲜血样可来源于骨髓、外周血及脐带血。
进一步地，造血干细胞的体外扩增方法，具体包括以下步骤：
S1：将丁酸钠溶解于无菌ddH 2O中，得到丁酸钠浓储溶液；
S2：将步骤S1中得到的丁酸钠浓储溶液添加到StemSpan SFEM II基本培养基中，得到细胞培养基；
S3：获取新鲜血样造血干细胞接种至步骤S2得到的细胞培养基中进行扩增培养，得到更大数量的造血干细胞。
进一步地，所述S1中所述丁酸钠的纯度≥98.5％，GC，所述丁酸钠浓储溶液的浓度为200mol/L，扩增时直接使用浓储溶液并根据样本体积进行稀释，浓储溶液须放置于-20℃保存。
进一步地，所述步骤S3进一步包括以下步骤：
S31：利用淋巴细胞分离液将新鲜血样分离得到单个核细胞，通过MACS分选得到CD34+细胞，即造血干细胞，随后将造血干细胞储存于液氮中；
S32：扩增培养前，先将造血干细胞解冻，加生理盐水重悬洗涤再离心，收集得到造血干细胞。
本发明的另一个方面，上述扩增造血干细胞的培养体系在以新鲜血样造血干细胞为样本细胞制备造血干细胞中的用途。
进一步地，所述步骤S3中的培养条件为：在37℃，CO2浓度为5％的条件下培养。
进一步地，所述步骤S3中的培养时间为7-10天。
本发明的有益效果：
1、通过添加丁酸钠显著提高了脐带血造血干细胞体外扩增所得的细胞总量，为更广泛的临床试验提供了前期技术基础；
2、扩增后的造血干细胞含有更高比例的CD34+CD90+细胞群，且具有更优异的CFU集落形成能力，表明这些造血干细胞更加原始，具有更强的 重建血液系统的分化潜能，可以更有效地支持临床治疗需要；
3、使得造血干细胞体外扩增更简便安全和高效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是各浓度丁酸钠作用下，脐带血造血干细胞各样本在第7天的细胞总数量和CD34+细胞总数量统计的结果图；
图2是各浓度丁酸钠作用下，脐带血造血干细胞各样本在第7天的CD34+CD45RA-和CD34+CD90+细胞总数量统计的结果图；
图3是各浓度丁酸钠作用下，脐带血造血干细胞代表性样本在第7天的CD34表面抗原表达情况分析图；
图4是各浓度丁酸钠作用下，脐带血造血干细胞代表性样本在第7天的CD34、CD45RA表面抗原表达情况分析图；
图5是各浓度丁酸钠作用下，脐带血造血干细胞代表性样本在第7天的CD34、CD90表面抗原表达情况分析图；
图6是倒置显微镜下各谱系集落形成代表图，其中a图为CFU-E；b图为CFU-G；c图为CFU-M；d图为CFU-GM；e图为CFU-GEMM；
图7是各浓度丁酸钠培养第7天脐带血造血干细胞样本各谱系集落形成单位数量分析图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员所获得的所有其他实施例，都属于本发明保护的范围。实施例中所使用的各种试剂及实验器材均可以从商业途径获得。
首先，对本发明实施例中出现的英文词汇及涉及的试剂材料作出说明：
StemSpan SFEM II是无血清培养基，生产厂商为StemCell  Technologies，货号为09655；
重组人干细胞因子rhSCF(recombined human stem cell factor)，生产厂商为Stemimmune LLC，货号为HHM-SF-1000；
重组人血小板生成素rhTPO(recombined human thrombopoietin)，生产厂商为Stemimmune LLC，货号为HHM-TP-0100；
重组人FMS样酪氨酸激酶3配体rhFLT3L(recombined human FMS-like tyrosine kinase 3 ligand)，生产厂商为Stemimmune LLC，货号为HHM-FT-1000；
丁酸钠，生产厂商为Sigma-Aldrich；
外周血单个核细胞PBMC(peripheral blood mononuclear cell)
MACS：磁珠分选；
DMSO：二甲亚砜；
PBS：磷酸盐缓冲液；
MethoCult TM GF H4435，是半固体培养基；
CFU-E全称Conoly Forming Unit of Erythrocyte，中文名为红细胞集落形成单位；
CFU-G全称Conoly Forming Unit of Granulocyte，中文名为粒细胞集落形成单位；
CFU-M全称Conoly Forming Unit of Macrophage，中文名为巨噬细胞集落形成单位；
CFU-GM全称Conoly Forming Unit of Granulocyte-Macrophage，中文名为粒细胞-巨噬细胞集落形成单位；
CFU-GEMM全称Conoly Forming Unit of granulocyte，erythrocyte，macrophage/monocyte，megakaryocyte，混合集落，其中文名为粒细胞，红细胞，巨噬/单核细胞，巨核细胞集落形成单位；
BFU-E全称Burst Forming Unit of Erythrocyte，中文名为爆发式红细胞集落形成单位。
实施例一
StemSpan SFEM II培养基可从StemCell Technologies公司购买，rhSCF， rhTPO，rhFLT3L购自Stemimmune LLC公司；尽管本发明的优先用于人类，其还可用于实验室动物，例如小鼠等；人类造血干细胞可来源于骨髓、外周血、脐带血和胎盘血，在本发明中，以脐带血造血干细胞为例，其中，脐带血采自健康产妇孕婴，经检测乙型肝炎、丙型肝炎、梅毒、艾滋病、巨细胞病毒、TORCH检测、支原体、衣原体、G-6PD和地贫均为阴性，人脐带血造血干细胞表达如下几种膜分子：白细胞分化抗原CD45、白细胞分化抗原CD34、白细胞分化抗原CD90、白细胞分化抗原CD49f。
1、获取外周血单个核细胞；
(1)用一次性血袋(含肝素钠等抗凝剂)采集脐带血80～120ml，将脐带血由血袋转移至500ml培养瓶中，加生理盐水稀释2～3倍，混匀后逐滴加入0.4倍体积淋巴细胞分离液中，注意不要破坏界面；
(2)使用1500～2000rpm/min离心20min，因密度不同离心管中由上到下分为四层：第一层为血浆层、第二层为环状乳白色单个核细胞层(PBMC)、第三层为透明分离液层、第四层为红细胞层；
(3)用吸管小心吸取第二层环状乳白色单个核细胞层(PBMC)到另一50ml离心管中，补加生理盐水，再次使用1500～2000rpm/min离心5～10min；
(4)弃上清加生理盐水重悬，最后使用1500～2000rpm/min离心5～10min，再次弃上清，得到PBMC细胞团块。
2、利用MACS从上述PBMC中获得CD34+脐带血造血干细胞；
(1)每份脐带血PBMC采用50ul人CD34+磁珠和50ul FcR blocker reagent及150ul 0.5％BSA的混合液重悬，4℃孵育30min；
(2)与此同时，将磁铁和磁力架至于超净台中紫外线照射30min；
(3)加入10ml无菌PBS，混匀后，使用1500～2000rpm/min离心5～10min后弃上清；
(4)将MACS专用吸附柱放进磁铁中，加入500ul 0.5％BSA润洗，流出的液体用15ml tube接住；
(5)500ul 0.5％BSA重悬获取外周血单个核细胞的步骤3)中PBMC团块，混匀后转移到MACS专用吸附柱中，待液体完全流出；
(6)500ul 0.5％BSA洗涤3次，取下吸附柱，置于15ml tube中；
(7)加入1ml 0.5％BSA，用活塞把液体推入15ml tube中，所得液体即含CD34+脐带血造血干细胞；
(8)稀释，计数，如要必要，用冻存保护剂DMSO将上述冻存于液氮中。
3、将所述CD34+脐带血造血干细胞悬浮接种于步骤2中得到的细胞培养基中进行培养，采用StemSpan SFEM II无血清培养基，添加SCF至SCF的浓度为80ng/ml，添加FLT3至FLT3的浓度为90ng/ml，添加TPO至TPO的浓度为30ng/ml；24孔板中细胞接种密度为1x10 4/孔，丁酸钠添加50μM，置于37℃，5％CO2培养箱培养。
4、根据细胞培养状态，每隔2天补加步骤2中得到的细胞培养基500μl，7～10天可获得数量较多的造血干细胞，扩增倍数约为4～20倍。
实施例二
与实施例一不同之处在于，将所述CD34+脐带血造血干细胞悬浮接种于步骤2中得到的细胞培养基中进行培养，采用StemSpan SFEM II无血清培养基，添加SCF至SCF的浓度为100ng/ml，添加FLT3至FLT3的浓度为100ng/ml，添加TPO至TPO的浓度为50ng/ml；24孔板中细胞接种密度为1x10 4/孔，丁酸钠添加100μM，置于37℃，5％CO2培养箱培养。
实施例三
与实施例一不同之处在于，将所述CD34+脐带血造血干细胞悬浮接种于步骤2中得到的细胞培养基中进行培养，采用StemSpan SFEM II无血清培养基，添加SCF至SCF的浓度为120ng/ml，添加FLT3至FLT3的浓度为110ng/ml，添加TPO至TPO的浓度为70ng/ml；24孔板中细胞接种密度为1x10 4/孔，丁酸钠添加200μM，置于37℃，5％CO2培养箱培养。
实施例四
实施例四为空白对照组，将所述CD34+脐带血造血干细胞悬浮接种于步骤2中得到的细胞培养基中进行培养，采用StemSpan SFEM II无血清培养基，添加SCF至SCF的浓度为80ng/ml，添加FLT3至FLT3的浓度为90ng/ml，添加TPO至TPO的浓度为30ng/ml；24孔板中细胞接种密度为1x10 4/孔，丁酸钠添加0μM，置于37℃，5％CO2培养箱培养。
实施例五 将上述实施例培养的脐带血造血干细胞进行表型鉴定及活率、纯度检测
1、细胞计数
分别对第7天的不同浓度的丁酸钠培养的细胞进行计数。
表1：CB CD34+细胞培养第7天各组条件细胞数目统计表
Figure PCTCN2018083216-appb-000001
2、细胞流式分析
分别对第0天、第7天的不同浓度的丁酸钠培养的CD34+细胞进行流式分析。采用BD公司FACS Verse流式检测仪，取细胞悬液20μl，加入溶解于0.5％BSA中的FITC标记的CD34，PE标记的CD38，APC-Cy7标记的CD45RA，APC标记的CD90。各管涡旋后室温下避光孵育15min，加入适量PBS，1600rpm室温水平离心5min，弃上清液，加入PBS 200μl，然后上机分析。
3、集落形成单位分析
分别对第0天、第7天的不同浓度的丁酸钠培养的CD34+细胞进行集落形成单位分析。采用MethoCult TM GF H4435半固体培养基，在六孔板中加入培养基1ml/孔，CD34+细胞接种密度为1000细胞/孔，置于37℃5％CO2培养箱培养14天后，计算各谱系集落数目，并拍摄照片。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 扩增造血干细胞的培养体系，包括细胞培养基，其特征在于，所述细胞培养基含有丁酸钠。
  2. 根据权利要求1所述的扩增造血干细胞的培养体系，其特征在于，所述细胞培养基中，丁酸钠的浓度为50～200μmol/L。
  3. 根据权利要求2所述的扩增造血干细胞的培养体系，其特征在于，所述细胞培养基中，丁酸钠的浓度为100μmol/L。
  4. 根据权利要求1所述的扩增造血干细胞的培养体系，其特征在于，所述细胞培养基还含有细胞因子，所述细胞因子为TPO、SCF、FLT3L中的至少。
  5. 根据权利要求4所述的扩增造血干细胞的培养体系，其特征在于，所述细胞培养基为含有丁酸钠、TPO、SCF和FLT3L的StemSpan SFEM II培养基。
  6. 根据权利要求4所述的扩增造血干细胞的培养体系，其特征在于，所述TPO在细胞培养基中的浓度为30-70ng/mL；所述SCF在细胞培养基中的为80-120ng/mL；所述FLT3L在细胞培养基中的浓度为90-110ng/mL。
  7. 造血干细胞的体外扩增方法，其特征在于，获取新鲜血样造血干细胞为样本细胞，采用权利要求1-6任一项所述的扩增造血干细胞的培养体系进行体外扩增培养，得到造血干细胞。
  8. 根据权利要求7所述的造血干细胞的体外扩增方法，其特征在于，包括以下步骤：
    S1：将丁酸钠溶解于无菌ddH 2O中，得到丁酸钠浓储溶液；
    S2：将步骤S1中得到的丁酸钠浓储溶液添加到StemSpan SFEM II基本培养基中，得到细胞培养基；
    S3：获取新鲜血样造血干细胞接种至步骤S2得到的细胞培养基中进行扩增培养，得到更大数量的造血干细胞。
  9. 根据权利要求8所述的造血干细胞的体外扩增方法，其特征在于，所述步骤S3进一步包括以下步骤：
    S31：利用淋巴细胞分离液将新鲜血样分离得到单个核细胞，通过MACS 分选得到CD34+细胞，即造血干细胞，随后将造血干细胞储存于液氮中；
    S32：扩增培养前，先将造血干细胞解冻，加生理盐水重悬洗涤再离心，收集得到造血干细胞。
  10. 权利要求1-6任一项所述的扩增造血干细胞的培养体系在以新鲜血样造血干细胞为样本细胞制备造血干细胞中的用途。
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