WO2003064636A1 - Separation, preparation and use of medullary hematopoietic stem cell - Google Patents
Separation, preparation and use of medullary hematopoietic stem cell Download PDFInfo
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- the present invention relates to a method for isolating, preparing and in vitro inducing a phenotype cell population with blood vascular stem cells. Background technique
- hematopoietic stem cell transplantation is used in the clinic to treat diseases such as blood and tumors, and vascular diseases caused by various causes have not been effectively treated.
- the main difficulty is that sufficient vascular stem cells cannot be obtained.
- many studies have shown that the presence of blood vascular stem cells in the yolk sac and embryonic stem cells can differentiate into hematopoietic and endothelial cells in both directions.
- implementation is difficult due to ethical and technical reasons. Therefore, it is important to isolate and culture blood vascular stem cells from human tissues.
- the present invention is to isolate, culture and identify a cell population with phenotypic characteristics of blood vessel stem cells from bone marrow cells, and provide a new therapeutic approach for certain diseases in the blood and blood vessels. Disclosure of invention
- the purpose of the present invention is to establish a method for isolating and culturing blood vascular stem cells from bone marrow for the first time in the world, and to provide a new way for the treatment of clinical diseases.
- the object of the present invention is achieved by the following methods:
- the induced cells were fixed with 4% paraformaldehyde together with the gel, and Wright stained. The distance between two adjacent cells was measured with a light microscope with a ruler.
- Lmg / ml of RNase A after cell cycle determination and cell growth mode After fetal bone marrow stromal cells were digested with trypsin, IX 10 5 cells / ml cells were permeated with 70% ethanol at 4 ° C for 10 minutes. Treat at 37 ° C for 20 minutes, then label the cell DNA with 3ug / ml propidium iodide for 5 minutes at room temperature, and place on ice for detection by flow cytometry.
- cells were trypsinized and seeded in 24-well plates with IX 10 4 cells per well. The cells were continuously cultured, and trypsin-digested viable cells were continuously counted every day with fetal blue, and the cell growth curve was drawn.
- Bone marrow stromal cells participate in angiogenesis after partial liver resection in rats To study whether bone marrow stromal cells participate in angiogenesis in vivo, we selected the characteristics of rat liver regeneration. Wister rats (purchased from the Fourth Academy of Military Medical Sciences), weighing about 250g, after intraperitoneal anesthesia with sodium pentobarbital, left liver resection was performed, about 40% of the liver was removed, and one day after liver resection, the tail vein was injected with 5 X 10 Five neonatal rat bone marrow stromal cells (cultured in the same way as fetal bone marrow stromal cells) at 1 day of birth were labeled with the fluorescent dye MPI. Three weeks after hepatectomy, frozen sections were taken from the liver and examined by fluorescence microscopy. '' Brief description of the drawings
- Figure 1 (a) is a growth curve of human fetal bone marrow stromal cells
- Figure 1 (b) is a cycle flow analysis diagram of human fetal bone marrow stromal cells
- Figure 2 shows the vascular-like structure when Flkl + cells differentiate into vascular endothelial cells for 11 days;
- Figure 3 is a blood vessel-like structure under an optical microscope
- Figure 4 (a) shows the shape of a transmission electron microscope in which slender endothelial cells form a vascular-like structure, with a gap made of gel in the middle (magnification 10,000 times);
- Figure 4 (b) shows the morphology of the ultrastructure of a single endothelial cell.
- the nucleus is slender spindle-shaped, and heterochromatin is visible;
- Figure 4 (c) shows the shape of a transmission electron microscope of a blood vessel-like structure where two adjacent cells are superimposedly connected
- Figure 5 (a) Immunohistochemical staining of CD34 positive round cells.
- FIG. 6 is a blood vessel-like structure diagram under a fluorescence microscope. The best way to implement the invention
- Adherently grown fetal bone marrow stromal cells have a typical fibroblast-like morphology, and the cell growth pattern is shown in [la].
- the growth curve shows that in the logarithmic growth phase, the cell doubling time is about 30 hours. 01
- the DNA content was detected by flow cytometry, and the cell cycle was analyzed (see Figure [lb]). About 89. 90% of the cells were in the G0 / G1 phase, while the cells in the G2-M phase and the S phase were 3.01. % And 7. 08%. So what we get is a rapidly proliferating cell population.
- Figure [3] shows a vascular-like structure composed of a plurality of elongated spindle-shaped cells arranged, and the nucleus is also elongated spindle-shaped.
- the transmission electron microscopy results are shown in Figure [4]:
- the tube-like structure is composed of elongated endothelial cells, and the gap is composed of gel in the middle. Two adjacent cells overlap and connect in the same manner as the endothelial cells that make up a blood vessel.
- the immunohistochemistry (5) showed that the spherical-like structure composed of round cells was the center.
- the tube-like structure was extended to the periphery, and the characteristic phenotype CD31 of cells constituting the tube-like structure was positive.
- CD34 is a characteristic feature of progenitor cells of hematopoietic and endothelial cells, but in this differentiation culture system, about 20% of cells express CD34 and are round cells. This result suggests that these round cells are hematopoietic cells, and the cells that make up the tube-like structure are vascular endothelial cells that induce differentiation.
- Rat livers have a strong ability to regenerate, and they can return to their original weight in 2-3 weeks after most of the liver has been removed. Three weeks after the liver was removed from the rat, the rat liver returned to its original weight and the liver lobes thickened.
- DAPI mainly binds to DNA bases and tubulin in cells. Frozen sections were found under a fluorescent microscope ( Figure [6]) in the central vein of the liver Endothelial cells have blue fluorescence. This indicates that bone marrow stromal cells can participate in the repair or formation of blood vessels in the environment of liver regeneration after liver resection.
- the isolated cells can form blood vessels in vitro and differentiate into hematopoietic cells.
- the isolated cells can participate in the formation of blood vessels in the body.
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Abstract
The invention disclosed a method of separation and preparation of medullary hematopoietic stem cell. The stem cell would differentiate into hematopoietic cell and endothelial cell in the vitro inducement system. The invention also disclosed a method of incubating hematopoietic stem cell. The inducement system of hematopoietic stem cell differentiating into hematopoietic cell and blood vessel and it's use of treating blood vessel diseases and tumors.
Description
骨髓血液血管干细胞分离、 制备和应用 Isolation, preparation and application of bone marrow blood vascular stem cells
技术领域 Technical field
本发明涉及具有血液血管干细胞表型细胞群体的分离、 制备及体外诱导方法。 背景技术 The present invention relates to a method for isolating, preparing and in vitro inducing a phenotype cell population with blood vascular stem cells. Background technique
目前， 临床用造血干细胞移植治疗血液和肿瘤等疾病， 而各种原因引起的血管性疾 病还没有有效的移植方法来治疗， 主要困难是不能获得足够的血管干细胞。 随着对血液 和血管研究的逐步深入， 多项研究表明在卵黄囊和胚胎干细胞存在血液血管干细胞， 可 以向造血和内皮细胞双向分化。 但由于伦理和技术方面的原因可实施存在困难。 所以从 人的组织中分离和培养血液血管干细胞十分重要。 本发明系从骨髓细胞分离、 培养和鉴 定具有血液血管干细胞表型特征的细胞群， 为血液和血管方面的某些疾病提供新的治疗 途径。 发明的公开 At present, hematopoietic stem cell transplantation is used in the clinic to treat diseases such as blood and tumors, and vascular diseases caused by various causes have not been effectively treated. The main difficulty is that sufficient vascular stem cells cannot be obtained. With the gradual deepening of research on blood and blood vessels, many studies have shown that the presence of blood vascular stem cells in the yolk sac and embryonic stem cells can differentiate into hematopoietic and endothelial cells in both directions. However, implementation is difficult due to ethical and technical reasons. Therefore, it is important to isolate and culture blood vascular stem cells from human tissues. The present invention is to isolate, culture and identify a cell population with phenotypic characteristics of blood vessel stem cells from bone marrow cells, and provide a new therapeutic approach for certain diseases in the blood and blood vessels. Disclosure of invention
本发明的目的是在国际上首次从骨髓中建立分离和培养血液血管千细胞的制备方 法， 为临床疾病的治疗提供新途径。 The purpose of the present invention is to establish a method for isolating and culturing blood vascular stem cells from bone marrow for the first time in the world, and to provide a new way for the treatment of clinical diseases.
本发明的目的是通过下述方法实现的： · · The object of the present invention is achieved by the following methods:
1. 分离和培养人骨髓基质来源的血液血管干细胞 1. Isolation and culture of blood vessel stem cells derived from human bone marrow stroma
取人骨髓细胞， 用密度为 1. 077g/cm3的 Ficoll分离液（天津血研所科技公司）分离 单个核细胞， 1200rpm离心 20分钟， 取白环以上的细胞 lOOOrpm离心 10分钟。 将离心 下的细胞打散并以 I X 107个 /ml细胞接种在 25cm2的培养瓶内， 培养液的成分为： 40%MCDB (Sigma), 2%胎牛血清 (Gibco), lOOu/ml 的青霉素和链霉素， DF12 (Gibco)。 在含 5%的 C02， 37°C培养箱内孵育。 第三天去掉悬浮细胞， 而贴壁细胞继续培养 6天左右， 每三天 换一次液。 细胞融合约 90%时， 用 0.25%的胰酶消化并以 1:1比例传代。 Human bone marrow cells were taken, and mononuclear cells were separated with Ficoll separation solution (Tianjin Blood Research Institute Technology Co., Ltd.) with a density of 1. 077 g / cm 3 , centrifuged at 1200 rpm for 20 minutes, and cells above the white ring were centrifuged at 1000 rpm for 10 minutes. The cells under centrifugation were dispersed and seeded in a 25 cm 2 culture flask with IX 10 7 cells / ml cells. The composition of the culture solution was: 40% MCDB (Sigma), 2% fetal bovine serum (Gibco), 100u / ml Penicillin and Streptomycin, DF 12 (Gibco). Were then incubated in C0 2, 37 ° C incubator containing 5% by weight. On the third day, the suspended cells were removed, while the adherent cells were cultured for about 6 days, and the solution was changed every three days. When the cells were about 90% confluent, they were digested with 0.25% trypsin and passaged at a 1: 1 ratio.
2. 人骨髓基质来源的血液血管干细胞在体外分化 2. Differentiation of human bone marrow stromal-derived blood vascular stem cells in vitro
基底膜胶 4°C包被 24孔板,每孔加 50ul,37°C聚合 20分钟。 为诱导胎儿骨髓基质来源 的血液血管干细胞的分化， 5 X 103个细胞接种在胶上进行三维体系培养。 分化培养液为 M199, 并含 VEGF, bFGF, FCS和 100u/ml 的青霉素和链霉素。 为了研究血管形成抑制 药苏拉明对血管形成的影响， 0. 01%的苏拉明分别在诱导后 1天和 8天加入诱导分化体系 中。
3. 血管样结构形态鉴定 Basement membrane gel coated 24-well plate at 4 ° C, add 50ul to each well, and polymerize at 37 ° C for 20 minutes. In order to induce the differentiation of fetal bone marrow stromal-derived blood vascular stem cells, 5 X 10 3 cells were seeded on a gel and cultured in a three-dimensional system. The differentiation medium was M199, and contained VEGF, bFGF, FCS and 100u / ml penicillin and streptomycin. In order to study the effect of the angiogenesis inhibitor suramin on angiogenesis, 0.01% of suramin was added to the induced differentiation system 1 day and 8 days after induction, respectively. 3. Identification of vascular-like structure
将诱导后的细胞与胶一起用 4%的多聚甲醛固定后， 瑞氏染色。 并用带有标尺的光学 显微镜测定两相邻细胞间的距离。 The induced cells were fixed with 4% paraformaldehyde together with the gel, and Wright stained. The distance between two adjacent cells was measured with a light microscope with a ruler.
细胞周期测定和细胞生长方式： 将胎儿骨髓基质细胞用胰酶消化后， 取 I X 105个 /ml 细胞用 70%的乙醇 4°C透膜 10分钟后， 用 0. lmg/ml的 RNase A 37°C处理 20分钟， 然后 室温下用 3ug/ml的碘化丙啶标记细胞的 DNA 5分钟， 置于冰上待流式细胞仪检测。 为研 究胎儿骨髓基质细胞的生长方式， 细胞胰酶消化以后接种在 24孔板， 每孔 I X 104个细胞。 细胞连续培养， 每天用胎盼兰连续计数胰酶消化的活细胞， 并画出细胞的生长曲线。 Lmg / ml of RNase A after cell cycle determination and cell growth mode: After fetal bone marrow stromal cells were digested with trypsin, IX 10 5 cells / ml cells were permeated with 70% ethanol at 4 ° C for 10 minutes. Treat at 37 ° C for 20 minutes, then label the cell DNA with 3ug / ml propidium iodide for 5 minutes at room temperature, and place on ice for detection by flow cytometry. To study the growth mode of fetal bone marrow stromal cells, cells were trypsinized and seeded in 24-well plates with IX 10 4 cells per well. The cells were continuously cultured, and trypsin-digested viable cells were continuously counted every day with fetal blue, and the cell growth curve was drawn.
血管样结构透射电镜鉴定： 把诱导后的细胞及胶块 6%的戊二醛固定 2-3小时后， 用 1%锇酸固定 2-3小时， 丙酮梯度脱水， 环氧丙烷、 812环氧树脂浸透， 60Ό聚合， 超薄切 片， 枸橼酸铅、 醋酸铀双染， 电镜观察。 Identification of vascular-like structures by transmission electron microscopy: After induction, 6% glutaraldehyde was fixed on the cells and gel blocks for 2-3 hours, then fixed with 1% osmic acid for 2-3 hours, acetone gradient dehydration, propylene oxide, 812 epoxy Resin soaked, polymerized at 60Ό, ultra-thin section, double stained with lead citrate and uranium acetate, observed by electron microscope.
骨髓基质细胞体内参与大鼠肝脏部分切除后血管的生成： 为研究骨髓基质细胞是否 在体内参与血管的生成， 我们选用了大鼠肝脏再生的特性。 Wister大鼠（购自军事医学 科学院四所），重约 250g，戊巴比妥钠腹腔麻醉后，行肝脏左中叶切除术，肝脏切除约 40%， 肝脏切除后一天， 尾静脉注射 5 X 105个刚出生 1天的乳鼠骨髓基质细胞（培养方法同胎 儿骨髓基质细胞）， 此细胞用荧光染料 MPI标记。 肝切除后 3 周， 取肝脏做冰冻切片， 荧光显微镜检测。 ' 附图的简要说明 Bone marrow stromal cells participate in angiogenesis after partial liver resection in rats: To study whether bone marrow stromal cells participate in angiogenesis in vivo, we selected the characteristics of rat liver regeneration. Wister rats (purchased from the Fourth Academy of Military Medical Sciences), weighing about 250g, after intraperitoneal anesthesia with sodium pentobarbital, left liver resection was performed, about 40% of the liver was removed, and one day after liver resection, the tail vein was injected with 5 X 10 Five neonatal rat bone marrow stromal cells (cultured in the same way as fetal bone marrow stromal cells) at 1 day of birth were labeled with the fluorescent dye MPI. Three weeks after hepatectomy, frozen sections were taken from the liver and examined by fluorescence microscopy. '' Brief description of the drawings
图 1 (a)为人胎儿骨髓基质细胞生长曲线图； Figure 1 (a) is a growth curve of human fetal bone marrow stromal cells;
图 1 (b)为人胎儿骨髓基质细胞周期流式分析图； Figure 1 (b) is a cycle flow analysis diagram of human fetal bone marrow stromal cells;
图 2为 Flkl+细胞向血管内皮细胞分化 11天时血管样的结构； Figure 2 shows the vascular-like structure when Flkl + cells differentiate into vascular endothelial cells for 11 days;
图 3为光学显微镜下的血管样结构； Figure 3 is a blood vessel-like structure under an optical microscope;
图 4 (a)为可见细长内皮的细胞组成血管样结构的透射电镜的形态,中间是由胶组成的 空隙 (放大 10000倍）； Figure 4 (a) shows the shape of a transmission electron microscope in which slender endothelial cells form a vascular-like structure, with a gap made of gel in the middle (magnification 10,000 times);
图 4 (b)为单个内皮细胞超微结构的透射电镜的形态,核为细长梭形，可见异染色质； 细胞浆可见内质网。 Figure 4 (b) shows the morphology of the ultrastructure of a single endothelial cell. The nucleus is slender spindle-shaped, and heterochromatin is visible;
图 4 (c)为两相邻细胞重叠连接的血管样结构的透射电镜的形态； Figure 4 (c) shows the shape of a transmission electron microscope of a blood vessel-like structure where two adjacent cells are superimposedly connected;
图 5 (a) CD34阳性的圆形细胞的免疫组化染色图； . Figure 5 (a) Immunohistochemical staining of CD34 positive round cells;.
图 5 (b) CD31阳性细胞的免疫组化染色图； Figure 5 (b) Immunohistochemical staining of CD31-positive cells;
图 6为荧光显微镜下血管样结构图。
实现本发明的最佳方式 FIG. 6 is a blood vessel-like structure diagram under a fluorescence microscope. The best way to implement the invention
分离和培养具有血液血管干细胞表型的应用例作 Application Examples for Isolating and Cultivating Hematopoietic Stem Cell Phenotypes
1. 人胎儿骨髓基质细胞的生物学特性 Biological characteristics of human fetal bone marrow stromal cells
贴壁生长的胎儿骨髓基质细胞呈典型成纤维样形态， 细胞生长方式如图 [la]生长曲线 显示： 在对数生长期， 细胞倍增时间约为 30 小时。 通过流式细胞仪检测 DNA含量， 分析细胞周期 （见图 [lb] )， 细胞约 89. 90%处在 G0/G1期， 而处在 G2-M期和 S期的细 胞分别为 3. 01%和 7. 08%。 所以我们获得的是快速增殖的细胞群。 Adherently grown fetal bone marrow stromal cells have a typical fibroblast-like morphology, and the cell growth pattern is shown in [la]. The growth curve shows that in the logarithmic growth phase, the cell doubling time is about 30 hours. 01 The DNA content was detected by flow cytometry, and the cell cycle was analyzed (see Figure [lb]). About 89. 90% of the cells were in the G0 / G1 phase, while the cells in the G2-M phase and the S phase were 3.01. % And 7. 08%. So what we get is a rapidly proliferating cell population.
2. 体外诱导血管样结构的形成过程 2. Formation of vascular-like structures induced in vitro
我们将胎儿骨髓基质细胞接种在 Gel胶上， 诱导后的 11天， 可见血管样的结构形成 彼此相连的网络结构如图 [2]。 We inoculated fetal bone marrow stromal cells on Gel gel, and 11 days after induction, it was seen that vascular-like structures formed a network structure connected to each other as shown in Figure [2].
将诱导 9天的细胞瑞氏染色后， 图 [3]可见多个细长梭型细胞排列组成的血管样结构， 核也为细长梭形。沿管样结构纵轴排列的两相邻细胞之间的距离为 109. 09±2. 43uni (mean 士 SEM， n=38)， 此结果与血管内皮细胞间的距离为 lOOum左右相符合。 After Wright staining of the cells induced for 9 days, Figure [3] shows a vascular-like structure composed of a plurality of elongated spindle-shaped cells arranged, and the nucleus is also elongated spindle-shaped. The distance between two adjacent cells arranged along the longitudinal axis of the tube-like structure was 109.09 ± 2. 43uni (mean ± SEM, n = 38). This result is consistent with the distance between vascular endothelial cells of about 100 μm.
3. 透射电镜鉴定细胞结构 3. Identification of cell structure by transmission electron microscopy
透射电镜的结果如图 [4]显示： 由细长内皮样的细胞组成管样结构， 中间是由胶组成 的空隙。 两相邻细胞重叠连接， 其连接方式与组成血管的内皮细胞的连接方式相同。 The transmission electron microscopy results are shown in Figure [4]: The tube-like structure is composed of elongated endothelial cells, and the gap is composed of gel in the middle. Two adjacent cells overlap and connect in the same manner as the endothelial cells that make up a blood vessel.
4. 免疫组化染色鉴定诱导的细胞免疫表型 4. Identification of induced cellular immune phenotype by immunohistochemical staining
为了鉴定组成血管样结构的细胞的免疫表型， 将诱导后有血管样结构的 Gel 胶块石 蜡包埋后， 免疫组化图（5)显示： 以圆形细胞组成的球样结构为中心， 向周围延伸出管样 结构， 组成管样结构的细胞特征性表型 CD31为阳性。 CD34是造血和内皮细胞的祖细胞的 表性特征， 但在此分化培养体系中约有 20%细胞表达 CD34, 且为圆形细胞。 所以此结果 提示这些圆形细胞是造血细胞， 而组成管样结构的细胞是诱导分化的血管内皮细胞。 In order to identify the immunophenotype of the cells that make up the vascular-like structure, after the gel gel block with the vascular-like structure was embedded in paraffin, the immunohistochemistry (5) showed that the spherical-like structure composed of round cells was the center. The tube-like structure was extended to the periphery, and the characteristic phenotype CD31 of cells constituting the tube-like structure was positive. CD34 is a characteristic feature of progenitor cells of hematopoietic and endothelial cells, but in this differentiation culture system, about 20% of cells express CD34 and are round cells. This result suggests that these round cells are hematopoietic cells, and the cells that make up the tube-like structure are vascular endothelial cells that induce differentiation.
5. 肝脏切除 5. Liver resection
大鼠肝脏再生能力很强， 在肝脏大部分切除后， 能在 2-3 周恢复到原来的重量。 我们 将大鼠肝切除后 3周， 大鼠肝脏恢复原重量， 肝叶增厚。 DAPI主要结合在 DNA碱基及 细胞内的微管蛋白。 冰冻切片后， 在荧光显微镜下发现 （图 [6] )， 在肝脏的中央静脉
内皮细胞处有蓝色荧光。 说明骨髓基质细胞在肝切除后肝脏再生的环境下， 能参与血 管的修复或形成。 Rat livers have a strong ability to regenerate, and they can return to their original weight in 2-3 weeks after most of the liver has been removed. Three weeks after the liver was removed from the rat, the rat liver returned to its original weight and the liver lobes thickened. DAPI mainly binds to DNA bases and tubulin in cells. Frozen sections were found under a fluorescent microscope (Figure [6]) in the central vein of the liver Endothelial cells have blue fluorescence. This indicates that bone marrow stromal cells can participate in the repair or formation of blood vessels in the environment of liver regeneration after liver resection.
本发明具有下列优点-The invention has the following advantages:
1 从人骨髓分离和培养血液血管干细胞。 1 Isolate and culture blood vascular stem cells from human bone marrow.
2 分离到的细胞在体外培养体系中能成血管， 并向造血细胞分化。 2 The isolated cells can form blood vessels in vitro and differentiate into hematopoietic cells.
3 分离到的细胞能在体内参与血管的形成。
3 The isolated cells can participate in the formation of blood vessels in the body.
Claims
1.一种制备具有血液血管干细胞表型细胞群的方法，其特征在于： 可向血管和造血细胞分 化的体外培养体系， 基底膜胶结合 VEGF和 bFGF的诱导体系。 What is claimed is: 1. A method for preparing a cell population having a vascular stem cell phenotype, comprising: an in vitro culture system that can differentiate into blood vessels and hematopoietic cells; and a basement membrane gel-binding VEGF and bFGF induction system.
2.根据权利要求 1 所述的方法， 其特征在于： 应用骨髓来源的血液血管干细胞， 用于人 工脏器血管构建的种子细胞。 The method according to claim 1, characterized in that: bone marrow-derived blood vascular stem cells are used as seed cells for constructing artificial organ blood vessels.
3.根据权利要求 1 所述的方法， 其特征在于： 应用骨髓来源的血液血管干细胞， 移植应 用于造血障碍性疾病及多种造血细胞肿瘤疾病的治疗的种子细胞。 The method according to claim 1, characterized in that: using bone marrow-derived blood vascular stem cells, transplanting seed cells that are used for the treatment of hematopoietic disorders and various hematopoietic tumor diseases.
4.根据权利要求 1 所述的方法， 其特征在于： 应用骨髓来源的血液血管干细胞， 移植应 用各种先天性及后天性心脏冠脉性疾病； 血管损伤， 先天及后天性动静脉痿、 血栓闭塞 性脉管炎、 动脉硬化性闭塞症、 血栓性静脉炎、 周围动脉瘤、 静脉曲张疾病的治疗及移 植构建人工血管的种子细胞。
The method according to claim 1, characterized in that: bone marrow-derived blood vascular stem cells are used for transplantation of various congenital and acquired cardiac coronary diseases; vascular injury, congenital and acquired arteriovenous loops, thrombi Treatment of occlusive vasculitis, arteriosclerotic occlusive disease, thrombophlebitis, peripheral aneurysm, varicose vein disease and transplantation of seed cells to construct artificial blood vessels.
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| JP2000325071A (en) * | 1999-05-21 | 2000-11-28 | Asahi Medical Co Ltd | Separation/recovery of cell |
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2000325071A (en) * | 1999-05-21 | 2000-11-28 | Asahi Medical Co Ltd | Separation/recovery of cell |
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
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