WO2016187820A1 - 一种人原代细胞培养基及其应用 - Google Patents

一种人原代细胞培养基及其应用 Download PDF

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WO2016187820A1
WO2016187820A1 PCT/CN2015/079846 CN2015079846W WO2016187820A1 WO 2016187820 A1 WO2016187820 A1 WO 2016187820A1 CN 2015079846 W CN2015079846 W CN 2015079846W WO 2016187820 A1 WO2016187820 A1 WO 2016187820A1
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medium
human
cells
culture
cell
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PCT/CN2015/079846
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French (fr)
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李鹏
蒋治武
林思妙
叶未
姚瑶
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中国科学院广州生物医药与健康研究院
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Priority to CN201580000125.7A priority Critical patent/CN105517672B/zh
Priority to PCT/CN2015/079846 priority patent/WO2016187820A1/zh
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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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  • the present invention relates to the field of cell culture technology, and in particular to a primary cell culture medium for the purpose of culturing human primary cells in vitro, and using the same to culture human primary acute B lymphocytic leukemia in vitro (B- ALL) Cell method.
  • B-cell acute lymphoblastic leukemia also known as pre-B-cell acute lymphoblastic leukemia
  • B-ALL is a malignant tumor derived from B-cell progenitor cells.
  • B-ALL is mainly a high-risk cancer in children, and its incidence in adults is decreasing.
  • the prognosis is good in B-ALL underage patients, and the long-term survival rate (EFS) is up to 90%, but it is a poor prognosis and low survival rate in B-ALL adults.
  • EFS long-term survival rate
  • traditional chemotherapy has a poor effect and the mortality rate is about 60%. Therefore, for B-ALL, new effective treatments need to be developed.
  • B-ALL related research mainly relies on the B-ALL cell line, and the cell line gradually adapts to the high concentration serum in the non-human environment during the long-term subculture, and forms some differences from the primary tumor cells. Gene mutations and cellular characteristics (such as high expression of p53 mutations).
  • patient-derived B-ALL sample cells can be expanded in vitro by xenografting into immunodeficient mice, in vivo experiments are expensive and time consuming, especially for new therapeutic methods or drug screening experiments. Material resources are unpredictable. Therefore, related studies on B-ALL require in vitro culture by means of primary B-ALL cells.
  • human primary cells are prone to differentiation and apoptosis when cultured in vitro; current human primary B-ALL cell culture methods are mainly cultured by serum culture medium supplemented with stromal cells and added with cytokines.
  • current human primary B-ALL cell culture methods are mainly cultured by serum culture medium supplemented with stromal cells and added with cytokines.
  • stromal cells OP9 were co-cultured with B progenitor cell line US7 or TXL2. Performed in ⁇ -MEM containing 20% FBS;
  • bone marrow mesenchymal stem BM-MSCs were co-cultured with B-lineage acute leukemia cells, and cytokines SCF, TPO were added. , FLT3L;
  • an article entitled "Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors” refers to an in vitro culture medium of primary lymphocytic leukemia cells, which is the addition of cytokines.
  • a serum-free IMDM medium of IL-3, IL-7, SCF although the medium does not contain serum, the inventors of the present invention performed repeated experiments using the medium, and found that the culture described in the article could not be achieved. The effect, that is, its stability needs to be studied;
  • WO 2013155405 A1 discloses a serum-free medium in which stromal cells are co-cultured with T-ALL, including EGF, hydrocortisone, insulin, SCF, IGF-1, IL-2 and IL-7.
  • serum is essential in the survival and culture of traditional cells in vitro
  • serum can accelerate the differentiation and aging of primary cells, especially hematopoietic stem cells and progenitor cells, when cultured in vitro in human primary cells;
  • the serum itself is concerned, even if the serum of the same brand, there may be significant differences between the serums of different batches, so that the culture effect is also significantly different, so the presence of the medium containing the serum-containing medium is not clear. The problem is relatively insufficient in practicality.
  • Co-culture with stromal cells is not only cumbersome, but also the growth rate of stromal cells (especially immortalized cell lines) far exceeds that of primary cells during co-culture, which competes with primary cells in the absorption medium.
  • Nutrition which in turn affects the growth of primary cells; in addition, stromal cells activate primary cells to survive and expand in vitro through protein secretion or receptor binding, however, these activation factors and cell status, differentiation, aging, and number of passages of stromal cells are not acceptable.
  • the control factors are related, which leads to the obvious difference in the culture effect of the primary cells cultured by the stromal cell co-culture method, which is difficult to be repeated and is not conducive to popularization and application.
  • the primary cell culture medium of the present invention contains no serum, and the desired in vitro culture effect can be achieved without co-culture of primary cells with stromal cells.
  • the invention provides a primary cell culture medium comprising a basal medium and a combination of cytokines; said cytokine combination comprising human FLT3L, human IGF1, human IL-7 and human IL-6.
  • the human FLT3L is contained in the medium in an amount of 5 to 100 ng/ml, preferably 25 to 100 ng/ml, more preferably 50 to 100 ng/ml;
  • the human FLT3L may be present in the medium in an amount of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml.
  • the human IGF1 is contained in the medium in an amount of 5 to 100 ng/ml, preferably 25 to 100 ng/ml, more preferably 50 to 100 ng/ml;
  • the human IGF1 may be present in the medium in an amount of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng/ml.
  • the human IL-7 is present in the medium in an amount of 2-50 ng/ml, preferably 10-50 ng/ml, more preferably 20-50 ng/ml;
  • the human IL-7 may be present in the medium in an amount of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 ng/ml.
  • the human IL-6 is present in the medium in an amount of 2-50 ng/ml, preferably 10-50 ng/ml, more preferably 20-50 ng/ml;
  • the human IL-6 may be present in the medium in an amount of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 ng/ml.
  • the medium further comprises any one or more of bovine serum albumin, transferrin and insulin.
  • the bovine serum albumin is contained in the medium in an amount of from 1 to 5 mg/ml, preferably from 2 to 4 mg/ml, more preferably 3 mg/ml;
  • the bovine serum albumin may be present in the medium in an amount of 1, 2, 3, 4 or 5 mg/ml.
  • the transferrin is contained in the medium in an amount of 1-20 ⁇ g/ml, preferably 5-15 ⁇ g/ml, more preferably 10 ⁇ g/ml;
  • the transferrin may be present in the medium in an amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20 ⁇ g/ml.
  • the content of the insulin in the medium is 1-20 ⁇ g / ml, preferably 5-15 ⁇ g / ml, more preferably 10 ⁇ g / ml;
  • the insulin may be present in the medium in an amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 ⁇ g/ml.
  • the medium comprises, in addition to the basal medium, the following components:
  • the basal medium is selected from the group consisting of IMDM, RPMI1640, ⁇ -MEM, and DMEM.
  • the invention provides the use of a primary cell culture medium according to the first aspect for culturing human primary cells in vitro.
  • the human primary cell is a human primary acute B lymphocyte leukemia (B-ALL) cell.
  • B-ALL human primary acute B lymphocyte leukemia
  • the present invention provides a method for in vitro culture of human primary acute B lymphocyte leukemia cells, comprising the step of in vitro culture using a primary cell culture medium as described in the first aspect.
  • the human primary cell culture medium of the present invention does not contain serum, and solves the problem of dependence of serum on the culture of human primary cells in vitro; and the medium avoids the co-culture of primary cells and stromal cells, and also solves the tradition. A series of problems with co-culture of stromal cells.
  • the in vitro culture using the medium of the present invention has higher amplification efficiency and longer incubation time than the existing culture method. For example, when used to culture and expand human primary B-ALL cells, the success rate of amplification is up to 90%, the total number of cells can be multiplied by more than 6 times, and the in vitro culture time can be as long as 2 months.
  • Figure 1 shows the success rate of in vitro culture of human primary B-ALL cells using the medium of the present invention
  • Figure 2 is a scattergram showing the cell viability of human primary B-ALL cells cultured in vitro using the medium of the present invention
  • Figure 3 is a bar graph showing cell viability of human primary B-ALL cells cultured in vitro using the medium of the present invention
  • Figure 4 shows cell growth curves of human primary B-ALL cells cultured in vitro using the medium of the present invention
  • Figure 5 is a graph showing the effect of the concentration of each cytokine in the medium of the present invention on the in vitro proliferation effect of human primary B-ALL cells;
  • Figure 6 shows the effect of cell proliferation of human primary B-ALL cells cultured in vitro using a medium containing a combination of different cytokines.
  • Example 1 evaluates the success rate of in vitro culture of human primary B-ALL cells using the medium of the present invention
  • lymphatic separation solution (Lymphoprep, StemCell Technologies) in a new 15 mL centrifuge tube;
  • primary B-ALL cells were obtained from bone marrow or peripheral blood samples of 20 patients with acute B lymphocytic leukemia, and a certain number of B-ALL cells were inoculated into the culture plate for one week in vitro. It is considered that the culture can be successfully cultured.
  • the culture medium containing serum but no combination of cytokines was used as a control, and the success rate of in vitro culture of human primary B-ALL cells using the medium of the present invention was examined.
  • the control medium composition is: IMDM + 10% FBS + 1% / P / S + 5 mM glutamate; the composition of the medium of the present invention is: IMDM + 1% / P / S + 5 mM glutamate + cytokine combination
  • the composition and content of the cytokine combination are as follows:
  • Fig. 1 The results are shown in Fig. 1.
  • the results in Fig. 1 show that 7 cases (35% of the total) were successfully cultured using the control medium; 18 cases (90% of the total) were successfully cultured using the medium of the present invention.
  • control medium composition is: IMDM + 10% FBS + 1% / P / S + 5 mM glutamate; the composition of the medium of the present invention is: IMDM + 1% / P / S + 5 mM glutamate + cytokine combination
  • the composition and content of the cytokine combination are as follows:
  • Human IGF1 100ng/ml Human IL-7 50ng/ml Human IL-6 50ng/ml
  • Example 3 Cell growth curve of human primary B-ALL cells cultured in vitro using the medium of the present invention
  • control medium composition is: IMDM + 10% FBS + 1% / P / S + 5 mM glutamate; the composition of the medium of the present invention is: IMDM + 1% / P / S + 5 mM glutamate + cytokine combination
  • the composition and content of the cytokine combination are as follows:
  • Fig. 4 shows that the leukemia cells were gradually reduced in the group using the control medium; the group using the medium of the present invention gradually increased in the number of cells as the number of culture days increased.
  • Example 4 In vitro culture time of human primary B-ALL cells cultured in vitro using the medium of the present invention
  • the control medium composition is: IMDM + 10% FBS + 1% / P / S + 5 mM glutamate; the composition of the medium of the present invention is: IMDM + 1% / P / S + 5 mM glutamate + cytokine combination
  • the composition and content of the cytokine combination are as follows:
  • Example 5 Assessing the in vitro proliferation of human primary B-ALL cells by the concentration of each cytokine in the medium of the present invention Effect of effect
  • control medium composition is: IMDM + 10% FBS + 1% / P / S + 5 mM glutamate; the composition of the medium of the present invention is: IMDM + 1% / P / S + 5 mM glutamate + cytokine combination
  • the composition and content of the cytokine combination are as follows:
  • the primary B-ALL cells prepared in Example 1 were seeded in a 96-well plate at a cell density of 1 ⁇ 10 5 /well. After 7 days, cell proliferation was detected using the Resazurin reduction method, and the greater the fluorescence intensity, the greater the number of cell proliferation.
  • the specific detection steps are:
  • Resazurin reagent corresponding to 10% of the volume of the medium in the well; for example, if the medium in the well is 100 ul, 10 ul of Resazurin reagent is added;
  • the culture time depends on the cell type and the number of cells; the plate can be read through multiple times in different time periods to determine the optimal time point;
  • Example 6 In vitro culture of human primary B-ALL cells using a medium containing a combination of different cytokines Cell proliferation effect
  • the cell proliferation effect of in vitro culture of human primary B-ALL cells using a medium containing a combination of different cytokines was evaluated using the cell proliferation assay method in Example 5.
  • the medium composition of each culture protocol was: IMDM + 1% P / S + 5 mM glutamate + cytokine combination, the cytokine combination of each culture protocol is shown in the following table, and the culture results are shown in Fig. 6.
  • cytokine combination i.e., FLT3L + IGF1 + IL-7 + IL-6
  • other cytokines are added to the culture medium of the present invention or one of the basic cytokine combinations is subtracted. Or certain cytokines can affect the proliferation of leukemia cells in vitro to varying degrees.

Abstract

提供了一种原代细胞培养基、该培养基用于体外培养人原代细胞的用途，以及使用该培养基体外培养人原代急性B淋巴细胞白血病(B-ALL)细胞的方法。所述原代细胞培养基除基础培养基外还包括细胞因子：人FLT3L、人IGFl、人IL-7和人IL-6，不含血清。

Description

一种人原代细胞培养基及其应用 技术领域
本发明涉及细胞培养技术领域，具体地，涉及一种原代细胞培养基，该培养基用于体外培养人原代细胞的用途，以及使用其体外培养人原代急性B淋巴细胞白血病(B-ALL)细胞的方法。
背景技术
急性B淋巴细胞白血病(B-ALL，B-cell acute lymphoblastic leukemia)，也称为前体B细胞(pre B-cell)急性淋巴细胞白血病，是来源于B细胞祖细胞的恶性肿瘤。B-ALL主要为儿童高发性癌症，在成人发病率下降。在B-ALL未成年患者中预后好，长期存活率(EFS，event-free survival)可达90％，但在B-ALL成人中却是预后差、低生存率。另外在成年B-ALL患者中，传统的化疗的效果差，死亡率约为60％。因此，针对B-ALL，需要研发新的有效治疗方法。
迄今为止，B-ALL相关研究主要依赖于B-ALL细胞系，而细胞系在长期传代培养的过程中，对高浓度血清的非人体环境中逐渐适应，并形成了一些区别于原代肿瘤细胞的基因突变和细胞特性(如高表达p53突变)。尽管病人来源的B-ALL样本细胞可以通过异种移植入免疫缺陷小鼠中扩大增殖后，进行体内实验，然而体内研究花费高、耗时长，特别是对于新型治疗方法或药物筛选实验，耗费的人力物力是不可预估的。因此，对于B-ALL的相关研究需要借助于原代B-ALL细胞的体外培养。
然而，人原代细胞在体外培养时容易分化、凋亡；目前的人原代B-ALL细胞培养方法主要是通过与基质细胞共培养、添加细胞因子的有血清培养基进行培养的。下面列举一些关于人原代B-ALL细胞培养的报道：
2015年3月的一篇名为“B-cell precursor acute lymphoblastic leukemia and stromal  cells communicate through Galectin-3”的文章中采用基质细胞OP9与B祖细胞白血病细胞系US7或TXL2共培养，所述共培养在含20％FBS的α-MEM中进行；
2014年12月份的一篇名为“mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells”的文章中将儿童B系白血病细胞与人骨髓基质细胞MSC共培养，所述共培养在含10％FBS和细胞因子SCF、IL-3、IL-7、FLT3L的基础培养基中进行；
一篇名为“Mesenchymal stem cells promote leukaemic cells aberrant phenotype from B-cell acute lymphoblastic leukaemia”的文章中将骨髓间充质干细BM-MSC与B系急性白血病细胞共培养，并加入细胞因子SCF、TPO、FLT3L；
2009年的一篇题为“Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors”的文章中提及了原代淋巴细胞白血病细胞的体外培养基，其为添加细胞因子IL-3、IL-7、SCF的无血清IMDM培养基；该培养基中虽然不包含血清，然而，本发明的发明人使用该培养基进行了重复实验，发现无法实现文章中所述的培养效果，即，其稳定性有待考究；
此外，WO 2013155405 A1中公开了基质细胞与T-ALL共培养的无血清培养基，该培养基包括EGF、氢化可的松、胰岛素、SCF、IGF-1、IL-2和IL-7等。
需要注意的是，血清虽然在传统细胞体外生存和培养过程中是必需的，然而，在人原代细胞体外培养时，血清可加速原代细胞特别是造血系干细胞、祖细胞的分化和老化；并且，就血清本身而言，即使是同一品牌的血清，不同批次的血清之间都可能存在明显差异，从而使得培养效果也具有明显差异，所以使用含血清的培养基存在培养基成分不明确的问题，实用性相对不足。
而与基质细胞共培养，不仅操作步骤麻烦，而且在共培养过程中，基质细胞(特别是永生化细胞系)的生长速度远超过原代细胞，其与原代细胞竞争吸收培养基中的 营养，进而影响原代细胞的生长；此外，基质细胞通过蛋白分泌或受体结合激活原代细胞体外存活和扩增，然而这些激活因素与基质细胞的细胞状态、分化、老化、传代次数等不可控因素相关，导致以基质细胞共培养方法培养的原代细胞的培养效果会出现明显差异，难以重复实施，不利于推广应用。
因此，本领域需要一种无需与基质细胞共培养的、不含血清的人原代细胞培养基。
发明内容
本发明的目的在于提供一种新的原代细胞培养基，该培养基用于体外培养人原代细胞的用途，以及使用其体外培养人原代B-ALL细胞的方法。本发明的原代细胞培养基不含血清，并且，无需原代细胞与基质细胞共培养，即可实现理想的体外培养效果。
本发明通过以下技术方案实现上述目的：
第一方面，本发明提供了一种原代细胞培养基，其包括基础培养基和细胞因子组合；所述细胞因子组合包括人FLT3L、人IGF1、人IL-7和人IL-6。
关于上述原代细胞培养基，作为优选，所述人FLT3L在培养基中的含量为5-100ng/ml、优选25-100ng/ml、更优选50-100ng/ml；
在具体实施方案中，所述人FLT3L在培养基中的含量可以为5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100ng/ml。
优选地，所述人IGF1在培养基中的含量为5-100ng/ml、优选25-100ng/ml、更优选50-100ng/ml；
在具体实施方案中，所述人IGF1在培养基中的含量可以为5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100ng/ml。
优选地，所述人IL-7在培养基中的含量为2-50ng/ml、优选10-50ng/ml、更优选20-50ng/ml；
在具体实施方案中，所述人IL-7在培养基中的含量可以为2、3、4、5、6、7、8、 9、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50ng/ml。
优选地，所述人IL-6在培养基中的含量为2-50ng/ml、优选10-50ng/ml、更优选20-50ng/ml；
在具体实施方案中，所述人IL-6在培养基中的含量可以为2、3、4、5、6、7、8、9、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50ng/ml。
作为优选，所述培养基还包括牛血清白蛋白、转铁蛋白和胰岛素中的任意一种或几种。
进一步优选地，所述牛血清白蛋白在培养基中的含量为1-5mg/ml、优选2-4mg/ml、更优选3mg/ml；
在具体实施方案中，所述牛血清白蛋白在培养基中的含量可以为1、2、3、4或5mg/ml。
优选地，所述转铁蛋白在培养基中的含量为1-20μg/ml，优选5-15μg/ml，更优选10μg/ml；
在具体实施方案中，所述转铁蛋白在培养基中的含量可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20μg/ml。
优选地，所述胰岛素在培养基中的含量为1-20μg/ml，优选5-15μg/ml，更优选10μg/ml；
在具体实施方案中，所述胰岛素在培养基中的含量可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20μg/ml。
在一个优选的具体实施方案中，除了基础培养基外，所述培养基包括如下含量的各组分：
Figure PCTCN2015079846-appb-000001
上述原代细胞培养基中，作为优选，所述基础培养基选自IMDM、RPMI1640、α-MEM和DMEM。
第二方面，本发明提供了如第一方面所述的原代细胞培养基在体外培养人原代细胞中的用途。
作为优选，所述人原代细胞为人原代急性B淋巴细胞白血病(B-ALL)细胞。
第三方面，本发明提供了一种人原代急性B淋巴细胞白血病细胞的体外培养方法，其包括使用如第一方面所述的原代细胞培养基进行体外培养的步骤。
有益效果
本发明的人原代细胞培养基不含血清，解决了人原代细胞体外培养对血清的依赖性问题；并且，所述培养基避免了原代细胞与基质细胞的共培养，也解决了传统的与基质细胞共培养所存在的一系列问题。此外，与现有培养方式相比，使用本发明培养基进行的体外培养具有更高的扩增效率，和更长的培养时间。例如，当用于培养、扩增人原代B-ALL细胞时，其扩增成功率可达90％，细胞数量总数可倍增达6倍以上，并且体外培养时间可长达2个月。
附图说明
图1显示使用本发明培养基体外培养人原代B-ALL细胞的成功率；
图2为散点图，显示使用本发明培养基体外培养人原代B-ALL细胞的细胞存活率；
图3为柱状图，显示使用本发明培养基体外培养人原代B-ALL细胞的细胞存活率；
图4显示使用本发明培养基体外培养人原代B-ALL细胞的细胞生长曲线；
图5显示本发明培养基中的各细胞因子浓度对人原代B-ALL细胞的体外增殖效果的影响；
图6显示使用包含不同的细胞因子组合的培养基，体外培养人原代B-ALL细胞的细胞增殖效果。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1评估使用本发明培养基体外培养人原代B-ALL细胞的成功率
通过密度离心法，分离获得人原代B-ALL细胞，具体步骤如下：
1)将B-ALL病人骨髓或外周血样本与生理盐水等比例混合；
2)在新的15mL离心管中加入稀释后血液体积二分之一的淋巴分离液(Lymphoprep，StemCell Technologies)；
3)将稀释后的血液沿离心管壁缓缓叠加于分层液面上，注意保持液面的清晰；
4)轻轻将骨髓样本混合物离心管放入离心机，800g/min离心20min；
5)轻轻取出离心管，骨髓样本混合物分层，用巴氏吸管小心吸取中间云雾层细胞置于新的15mL离心管；
6)用无血清IMEM培养基或PBS清洗两次，分离出的细胞用培养基重悬即为人原代B-ALL细胞样本。
按上述方法，从20例急性B淋巴细胞白血病患者的骨髓或外周血样品中，获得原代B-ALL细胞，将一定数量的B-ALL细胞接种于培养板中，能在体外培养一周以 上且细胞能增殖的视为培养成功。
本实施例以含血清但不含各细胞因子组合的培养基为对照，检测了使用本发明培养基体外培养人原代B-ALL细胞的成功率。
对照培养基组成为：IMDM+10％FBS+1％/P/S+5mM谷氨酸盐；本发明培养基组成为：IMDM+1％/P/S+5mM谷氨酸盐+细胞因子组合，所述细胞因子组合的成分及含量见下表：
成分 浓度
BSA 5mg/ml
转铁蛋白 5ug/ml
胰岛素 5ug/ml
人FLT3L 50ng/ml
人IGF1 50ng/ml
人IL-7 20ng/ml
人IL-6 20ng/ml
结果如图1所示，图1结果表明：使用对照培养基，培养成功7例(占总数的35％)；使用本发明培养基，培养成功18例(占总数的90％)。
实施例2评估使用本发明培养基体外培养人原代B-ALL细胞的细胞存活率
使用实施例1中制备的人原代B-ALL细胞样本，以含血清但不含细胞因子组合的对照培养基为对照，评估使用本发明培养基体外培养人原代B-ALL细胞的细胞存活率。对照培养基组成为：IMDM+10％FBS+1％/P/S+5mM谷氨酸盐；本发明培养基组成为：IMDM+1％/P/S+5mM谷氨酸盐+细胞因子组合，所述细胞因子组合的成分及含量见下表：
成分 浓度
BSA 1mg/ml
转铁蛋白 10ug/ml
胰岛素 10ug/ml
人FLT3L 100ng/ml
人IGF1 100ng/ml
人IL-7 50ng/ml
人IL-6 50ng/ml
具体地，将一定数量的人原代B-ALL细胞接种于培养板中，分别在第3天和第7天检测活细胞占总细胞数的比例。
结果如2和图3所示，图2和图3结果显示：与对照培养基相比，本发明培养基显著提高人原代B-ALL细胞的体外存活率。
实施例3使用本发明培养基体外培养人原代B-ALL细胞的细胞生长曲线
使用实施例1中制备的人原代B-ALL细胞样本，以含血清但不含细胞因子组合的对照培养基为对照，评估使用本发明培养基体外培养人原代B-ALL细胞的细胞生长状况。对照培养基组成为：IMDM+10％FBS+1％/P/S+5mM谷氨酸盐；本发明培养基组成为：IMDM+1％/P/S+5mM谷氨酸盐+细胞因子组合，所述细胞因子组合的成分及含量见下表：
成分 浓度
BSA 5mg/ml
5 5ug/ml
胰岛素 5ug/ml
人FLT3L 50ng/ml
人IGF1 50ng/ml
人IL-7 20ng/ml
人IL-6 20ng/ml
具体地，将5×105个该原代B-ALL细胞接种于24孔板中，培养基为1mL/孔，每3天半换一次新鲜培养基，每7天按初始细胞浓度进行细胞传代并计数，实验重复3次，绘制细胞生长曲线。
结果如图4所示，图4显示：使用对照培养基的组，白血病细胞逐渐减少；使用本发明培养基的组，随着培养天数增长，细胞数目逐渐增加。
实施例4使用本发明培养基体外培养人原代B-ALL细胞的体外培养时间
使用实施例1中制备的人原代B-ALL细胞样本，以含血清但不含细胞因子组合的对照培养基为对照，评估使用本发明培养基体外培养人原代B-ALL细胞的体外培养时间。对照培养基组成为：IMDM+10％FBS+1％/P/S+5mM谷氨酸盐；本发明培养基组成为：IMDM+1％/P/S+5mM谷氨酸盐+细胞因子组合，所述细胞因子组合的成分及含量见下表：
成分 浓度
BSA 5mg/ml
转铁蛋白 5ug/ml
胰岛素 5ug/ml
人FLT3L 50ng/ml
人IGF1 50ng/ml
人IL-7 20ng/ml
人IL-6 20ng/ml
结果如下表所示：
Figure PCTCN2015079846-appb-000002
上表结果表明，与对照培养基相比，本发明培养基的体外培养时间延长。
实施例5评估本发明培养基中的各细胞因子浓度对人原代B-ALL细胞的体外增殖 效果的影响
使用实施例1中制备的人原代B-ALL细胞样本，以含血清但不含细胞因子组合的对照培养基为对照，评估含不同浓度细胞因子的本发明培养基对人原代B-ALL细胞的体外增殖效果的影响。对照培养基组成为：IMDM+10％FBS+1％/P/S+5mM谷氨酸盐；本发明培养基组成为：IMDM+1％/P/S+5mM谷氨酸盐+细胞因子组合，所述细胞因子组合的成分及含量见下表：
  细胞因子组合1 细胞因子组合2 细胞因子组合3 细胞因子组合4
人FLT3L 5ng/ml 25ng/ml 50ng/ml 100ng/ml
人IGF1 5ng/ml 25ng/ml 50ng/ml 100ng/ml
人IL-7 2ng/ml 10ng/ml 20ng/ml 50ng/ml
人IL-6 2ng/ml 10ng/ml 20ng/ml 50ng/ml
具体地，将实施例1中所制备的原代B-ALL细胞接种于96孔板中，细胞密度为1×105/孔。7天后，使用Resazurin还原方法检测细胞增殖，荧光强度越大表示细胞增殖数量越多。具体检测步骤为：
1)解冻Resazurin试剂，并置于37度确保所有成分都溶解；
2)将原代B-ALL细胞接种于96孔板(孔间不透光，底部透明)，种板细胞浓度一般为1×105细胞/孔，细胞浓度主要取决于细胞生长速度；
3)分别加入对照培养基与本发明培养基，培养原代B-ALL细胞1-72小时，确保培养过程中，各孔的细胞培养基体积一样；
4)加入相当于孔内培养基体积10％的Resazurin试剂；比如，孔内培养基为100ul，则加入10ul的Resazurin试剂；
5)在正常培养条件培养1-6小时，培养时间取决于细胞类型和细胞数；可通过不同时段的多次读板以确定最佳时间点；
6)分别读取各孔在570nm波长下激发的590nm波长的荧光强度，最后求得相对荧光值RFU(Relative Fluorescent Units)。RFU值越高，表示细胞数越多。
结果如图5所示，图5结果表明：FLT3L、IGF1、IL-7、IL-6四种细胞因子含量越高，细胞增殖效果越明显。
实施例6使用包含不同的细胞因子组合的培养基，体外培养人原代B-ALL细胞 的细胞增殖效果
使用实施例5中的细胞增殖试验方法，评估使用包含不同的细胞因子组合的培养基体外培养人原代B-ALL细胞的细胞增殖效果。各培养方案的培养基组成为：IMDM+1％P/S+5mM谷氨酸盐+细胞因子组合，各培养方案的细胞因子组合见下表，培养结果如图6所示。
Figure PCTCN2015079846-appb-000003
Figure PCTCN2015079846-appb-000004
由图6可知，除了基础细胞因子组合(即FLT3L+IGF1+IL-7+IL-6)外，在本发明的培养基中添加其它细胞因子或者减去所述基础细胞因子组合中的某个或某些细胞因子可以不同程度的影响白血病细胞体外增殖效果。
申请人声明，本发明通过上述实施例来说明本发明的产品、用途及其使用方式，但本发明并不局限于上述详细用途和使用方式，即不意味着本发明必须依赖上述详细用途和使用方式才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (9)

  1. 一种原代细胞培养基，其包括基础培养基和细胞因子组合；所述细胞因子组合包括人FLT3L、人IGF1、人IL-7和人IL-6。
  2. 根据权利要求1所述的培养基，其特征在于，所述人FLT3L在培养基中的含量为5-100ng/ml、优选25-100ng/ml、更优选50-100ng/ml；
    优选地，所述人IGF1在培养基中的含量为5-100ng/ml、优选25-100ng/ml、更优选50-100ng/ml；
    优选地，所述人IL-7在培养基中的含量为2-50ng/ml、优选10-50ng/ml、更优选20-50ng/ml；
    优选地，所述人IL-6在培养基中的含量为2-50ng/ml、优选10-50ng/ml、更优选20-50ng/ml。
  3. 根据权利要求1或2所述的培养基，其特征在于，所述培养基还包括牛血清白蛋白、转铁蛋白和胰岛素中的任意一种或几种。
  4. 根据权利要求3所述的培养基，其特征在于，所述牛血清白蛋白在培养基中的含量为1-5mg/ml、优选2-4mg/ml、更优选3mg/ml；
    优选地，所述转铁蛋白在培养基中的含量为1-20μg/ml，优选5-15μg/ml，更优选10μg/ml；
    优选地，所述胰岛素在培养基中的含量为1-20μg/ml，优选5-15μg/ml，更优选10μg/ml。
  5. 根据权利要求1-4任一项所述的培养基，其特征在于，除了基础培养基外，所述培养基包括如下含量的各组分：
    Figure PCTCN2015079846-appb-100001
    Figure PCTCN2015079846-appb-100002
  6. 根据权利要求1-5任一项所述的培养基，其特征在于，所述基础培养基选自IMDM、RPMI1640、α-MEM和DMEM。
  7. 根据权利要求1-6任一项所述的培养基在体外培养人原代细胞中的用途。
  8. 根据权利要求7所述的用途，其特征在于，所述人原代细胞为人原代急性B淋巴细胞白血病细胞。
  9. 一种人原代急性B淋巴细胞白血病细胞的体外培养方法，其包括使用权利要求1-6任一项所述的培养基进行体外培养的步骤。
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