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CN103305453A - Microcarrier culture system of umbilical cord mesenchymal stem cells - Google Patents

Microcarrier culture system of umbilical cord mesenchymal stem cells Download PDF

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CN103305453A
CN103305453A CN 201310193663 CN201310193663A CN103305453A CN 103305453 A CN103305453 A CN 103305453A CN 201310193663 CN201310193663 CN 201310193663 CN 201310193663 A CN201310193663 A CN 201310193663A CN 103305453 A CN103305453 A CN 103305453A
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msc
culture
microcarrier
system
cells
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CN 201310193663
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侯亚义
赵树立
曹志刚
米琼宇
樊竤冶
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侯亚义
赵树立
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Abstract

The invention belongs to the field of biological medicines, and discloses a microcarrier culture system of umbilical cord mesenchymal stem cells (MSC), and preparation and applications of an MSC preserving fluid. The microcarrier culture system is characterized by preparing a heparin crosslinking sephadex microcarrier and a mating microcarrier culture medium, and a high-efficiency MSC culture system can be built. MSC cells can be efficiently and fast prepared within a short period by utilizing the microcarrier culture system of the MSC. The MSC preserving fluid is characterized by adding human albumin, heparin sodium, estrogen and IL-1beta into a ringer solution, the cell activity of the MSC cells can be kept unchanged within 24 hours by utilizing the preserving fluid, and the level of cell factors can be improved by about five times on average. The microcarrier culture system is applied to being developed into large-batch preparation of umbilical cord MSC for clinical use and laboratories, and provides a specific preserving method for MSC for treating systemic lupus erythematosus, and establishes a basis for the clinical application of the MSC.

Description

一种脐带间充质干细胞的微载体培养系统 UCB inter-microcarrier culture system of mesenchymal stem cells

技术领域 FIELD

[0001] 本发明是一种快速、有效和规模化获得脐带MSC的方法,具体地说是利用细胞微载体培养技术和特殊细胞因子作为培养体系而构成的无血清、规模化的提高获得脐带间充质干细胞得率、速度和质量,并根据不同的临床运用而开发出特殊的细胞保存液。 [0001] The present invention is a fast, effective, and umbilical cord obtained MSC scale process, particularly the use of a serum-free cell culture microcarrier technology and specific cytokine culture system constituted as to improve the scale of the umbilical cord between the obtained mesenchymal stem cells yield, speed and quality, and the development of particular cell storage solution depending on the clinical application.

技术背景 technical background

[0002] 间充质干细胞(MSC)是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中,其除了具有干细胞的多向分化潜能,还具有免疫调节功能、低免疫原性和易获取性等特点,目前已经受到临床上的广泛重视。 [0002] Mesenchymal stem cells (MSC) belonging mesoderm a class of pluripotent stem cells, primarily in the connective tissue and organ stroma, which in addition to differentiation potential of stem cells, also having immune function, low immunogenicity immunogenicity and accessibility features, has been widely importance clinically. 近年来MSC已成为干细胞研究中的热点,在免疫疾病治疗、造血干细胞移植、组织工程、基因工程等领域具有较好的应用前景。 In recent years, MSC has become the focus of stem cell research in the treatment of immune disorders, hematopoietic stem cell transplantation, tissue engineering, genetic engineering has good application prospects. 但MSC的临床运用受到其获取的数量、质量和定向分化等技术方面的限制,目前还没有进行广泛的临床应用。 However, the clinical use of MSC limited number of technical aspects of its acquisition, quality and directed differentiation, there is no extensive clinical application. 因此建立稳定有效快速的MSC规模化培养系统具有极为重要的意义。 It has very important significance to establish a stable, effective and rapid scale of MSC culture systems.

[0003] 一般认为MSCs在骨髓组织中的含量最丰富,但是随着年龄的老化,自体骨髓中MSCs数目显著降低、增殖分化能力大幅度衰退,目前从脂肪、胚胎、羊膜液、胎盘、脐带血以及脐带等骨髓以外的组织中分离出MSC已经成为研究热点。 [0003] MSCs in the bone marrow is generally believed that the content of the most abundant tissues, but with age, aging, the number of autologous bone marrow MSCs significantly reduced, greatly decline proliferation and differentiation, the present fat, embryo, amniotic fluid, placenta, umbilical cord blood and tissue other than the bone marrow like umbilical cord isolated MSC has become a hot topic. 其中脐带由于其易于获取,且脐带MSC细胞和骨髓MSC细胞具有相同的表型和分化潜能。 Because of their ease in which the umbilical cord acquisition, MSC cells and bone marrow and umbilical cord MSC cells with the same phenotype and differentiation potential.

[0004]目前用于分离间充质干细胞的方法主要有4种,即流式细胞分选法、磁珠分离法、密度梯度离心法和贴壁筛选法。 [0004] Current methods for the separation between the mesenchymal stem cells there are four kinds, i.e., flow cytometric sorting, magnetic bead separation, density gradient centrifugation and adherence screening method. 其中,贴壁筛选法,所获得的细胞形态均一,增殖速度快,生长状态稳定,且操作简便,成本低,有望成为较实用的生产方法。 Wherein adherence screening method, the cells obtained were highly homogeneous, fast growth rate, growth state stability, and easy to operate, low cost, is expected to become more practical production methods. 理论上MSC具有无限的增殖能力,但由于体外培养时MSC的·未分化状态只能维持有限的时间,而且随着传代次数的增殖,MSC的增殖能力下降,形态也由长梭形变成宽大扁平,逐渐丧失多向分化的能力。 MSC theoretically unlimited proliferative capacity, but due to the MSC-vitro undifferentiated state can only be maintained for a limited time, with the proliferation and passage number, MSC proliferative capacity decreased, but also into a long spindle form large flat, multi gradual loss of the ability to differentiate. 贴壁生长由于受到培养容器的限制,需要多次胰酶的消化、传代和扩增,过程繁琐,容易污染,而且营养物和细胞代谢产物导致的培养环境不均一,不利于MSC的大规模培养。 Since the growth of adherent culture vessel is limited, requiring multiple trypsin digestion, and passaged amplification process cumbersome, easily contaminated, and nutrients and metabolites cause cell culture environment uneven, the MSC is not conducive to large scale culture .

[0005]目前好多研究表明,一些细胞因子可以促进细胞的增殖,如酸性成纤维细胞因子、肝素、VEGF和FGF-2等等。 [0005] There is currently a lot of research suggests that some cytokine may promote cell proliferation, such as acidic fibroblast growth factor, heparin, VEGF and FGF-2 and the like. 虽然,这些细胞因子均可以不同程度的增加的MSC的增殖能力,但其作用机制尚未完全阐明,对MSC的增殖和分化影响十分复杂,这给临床应用安全性评估带来困难。 Although these cytokines can be increased to varying degrees of MSC proliferative capacity, but its mechanism is not fully understood, proliferation and differentiation of MSC's very complicated, which gives clinical safety assessment difficult. 为了增加MSC的增殖能力,而加入后不考虑其分化潜能违背了MSC的临床意义。 To increase the MSC proliferative capacity, without regard to their differentiation potential after joining violated the clinical significance of the MSC.

[0006] 1967年,Van Wezel率先用离子交换树脂DEAE-SephadexA-50制成微载体,用于培养细胞。 [0006] In 1967, Van Wezel lead with an ion exchange resin DEAE-SephadexA-50 made microcarrier for culturing cells. 微载体培养与单层细胞培养相比,为贴壁细胞提供了更大的培养表面积,从而使细胞的产量大大增加,而且易于控制,减少了污染的危险,使大规模生产成为可能。 Microcarrier culture as compared with monolayer cell culture, the culture provides a greater surface area of ​​the adherent cells, such that the significantly increased cell yield, and easy to control, reduce the risk of contamination, making mass production possible. 目前,采用微载体细胞培养系统在搅拌生物反应器内培养人的MSC细胞,以探索一种大量、快速的MSC培养方法已经成为一种趋势。 Currently, the use of microcarrier cell culture system of human MSC cells cultured in a stirred bioreactor to explore a large, rapid method for the MSC culture has become a trend. 微载体是直径在60〜250 μ m的微珠。 Microcarrier beads in diameter of 60~250 μ m. 目前,还没有适用于MSC大规模培养的微载体培养系统及其专用培养基,多采用培养其它细胞的培养系统和一般的含有动物源性血清的普通培养基,这导致了以下几个问题:①凋亡细胞较多:这主要是由于常用的微载体系统在普通的旋转式生物反应器搅拌情况下产生的剪切力较大,对MSC细胞造成一定损伤;②收集细胞较为困难:由于MSC是贴壁生长的细胞,所以在普通的微载体培养体系下收集细胞时候需要用0.25%胰酶进行消化收集,而一些多孔的微载体培养的MSC收集就更为困难;③异源性蛋白:在一般培养条件下在培养基中均加有10%的胎牛血清。 At present, no suitable system and a microcarrier culture medium dedicated MSC large scale cultivation, the use of cell culture, and other culture systems an ordinary medium containing animal serum, which leads to the following questions: ① more apoptotic cells: this is mainly due to the conventional microcarrier system was stirred larger shearing force generated in the case of an ordinary rotary bioreactor, cause some damage to the MSC cells; ② it is difficult to collect the cells: Since the MSC is attached cell wall growth, thus collected under ordinary microcarrier cell culture system when required digestion with 0.25% trypsin to collect, while some porous microcarrier culture is more difficult to collect the MSC; ③ heterologous protein: under normal culture conditions in a culture medium are supplemented with 10% fetal calf serum. 2010年,美国、欧盟等发达国家已全面停止在生物制药中应用血清。 In 2010, the United States, the European Union and other developed countries has been fully stopped using serum in biopharmaceuticals. 因此,在微载体细胞培养中采用无血清、无蛋白和化学成分限定培养基,成为细胞培养的研究热点。 Thus, in microcarrier cell cultures in serum-free, protein-free defined medium and chemical composition, cell culture has become a hot research.

[0007] 我们改进了传统的微载体培养体系,采用了新材料的微载体介质,并运用波浪式生物反应器将少了传统培养过程剪切力对细胞的损伤,并且采用了微载体培养特制的无血清培养基,将少了在MSC收集过程中的细胞损失,极大的提高了MSC的得率和效率。 [0007] We have improved the conventional microcarrier culture system, using a micro-carrier medium of new materials and the use of the wave bioreactor cultivation process less conventional shear damage to cells, and uses a special microcarrier culture the serum-free medium, the cell loss in the less MSC collection process and greatly improves the efficiency and yield of the MSC. 本方法简单易行,更有利于用于生产实践。 This method is simple, more conducive for production practice.

发明内容 SUMMARY

[0008] 本发明的目的是提供一种微载体培养系统,以快速有效利用新生胎儿的脐带为资源获取脐带MSC以及快速MSC鉴定的方法。 [0008] The object of the present invention is to provide a microcarrier culture system to quickly and efficiently acquire new born fetal umbilical cord MSC and a method for the rapid identification of MSC resources. 这套微载体培养系统具体包括如下内容: This microcarrier culture system comprises the following:

[0009] 1、肝素交联葡聚糖微载体:在传统的Cytodex3微载体制备过程中,增加按照变性胶原:肝素(10: I比例)混合与交联葡聚糖基架化学偶联。 [0009] 1, heparin cross-linked dextran microcarriers: Cytodex3 preparation in conventional microcarrier, increased by a denatured collagen: heparin (10: I ratio) was mixed with cross-linked dextran pedestal chemical coupling. 变性胶原层易被胰蛋白酶和胶原酶等多种蛋白酶消化,以便在用胰酶收集细胞时,能在维持细胞最大活性、功能和完整性的同时将细胞从微载体上分离出来。 Denatured collagen layer is susceptible to trypsin and other proteases and collagenase digestion, so that when the cells were collected with trypsin, the cells can be maintained in the maximum activity, function and integrity while separating the cells from the microcarriers. 在微载体表面的肝素可以促进MSC细胞的增殖与生长。 Heparin microcarrier surface may promote the proliferation and growth of MSC cells.

[0010] 2、MSC细胞无血清微载体培养基:其主要成分为AM-V培养基,里面加入人FGF-2 (50ng/ml),人Midkine (60ng/ml),大豆胰酶抑制剂(5ng/ml),青霉素(100units/ml)和链霉素(100units/ml)。 [0010] 2, MSC microcarrier cell culture medium without serum: The main components of the medium AM-V, which was added human FGF-2 (50ng / ml), human Midkine (60ng / ml), soybean trypsin inhibitor ( 5ng / ml), penicillin (100units / ml) and streptomycin (100units / ml).

[0011] 3、MSC细胞无血清微载体培养条件:微载体密度为3g/l肝素交联葡聚糖微载体,接种密度为0.3X106细胞/ml,接种在Cellbag-1L的生物反应器中,以IOrpm,6°,Ph7.0-7.2,37°C,5% C02 为培养条件。 [0011] 3, MSC microcarrier cell culture serum-free conditions: microcarrier density of 3g / l heparin cross-linked dextran microcarrier inoculation density of 0.3X106 cells / ml, seeded in a Cellbag-1L bioreactor, in IOrpm, 6 °, Ph7.0-7.2,37 ° C, 5% C02 for the culture conditions.

[0012] 4、治疗用MSC保存液:该保存液主要成分为临床用林格氏液,I %的临床用人白蛋白和1000AxaIU/ml的临床用肝素纳。 [0012] 4, treatment with MSC preservation solution: the solution is saved as the main component of a clinical Ringer's solution, I% human albumin and clinical 1000AxaIU / ml of sodium heparin for clinical use. 然后根据MSC在不同疾病中发挥的不同作用加入特殊的细胞因子以维护或加强MSC的生物活性。 The MSC then play different roles in different diseases particular cytokines was added to maintain or enhance the biological activity of the MSC. 系统性红斑狼疮治疗用MSC保存液(上述保存液,加有10_7雌激素和50ng/ml的IL-1 ^ ),心脑血管治疗用MSC保存液(上述保存液,加有50ng/ml的人Midkine)和成骨治疗用MSC保存液(上述保存液,加有人骨形成蛋白BMP100ng/ml)。 Systemic treatment with MSC preservation solution (the aqueous fluid, and estrogen plus 10_7 of 50ng / ml IL-1 ^), cardiovascular treatment preservation solution (the aqueous fluid, plus 50ng / ml with human MSC midkine) and treated with osteogenic MSC storage solution (storage solution described above, was added to bone morphogenetic protein BMP100ng / ml).

附图说明: BRIEF DESCRIPTION OF:

[0013] 图1、MSC得率的比较:相同体积培养基在不同培养体系下得到的MSC细胞的数量和时间的比较。 Comparison MSC yield [0013] FIG 1: Comparison of the number and time of the same volume of medium MSC cells obtained in different culture systems. 在10天的培养微载体培养系统条件下一共用了IL的培养基,采用传统的贴壁培养方法可以得到1.56X 108个细胞(图1,A),而采用传统的微载体培养方法,用IL含血清的细胞培养基可以得到3.17 X IO9个细胞(图1,B),而采用本发明的微载体无血清培养系统,IL培养基可以得到5.78 X IO9个细胞(图1,C)。 10-day culture in the common system conditions microcarrier culture medium of IL Next, the conventional method of culturing adherent cells may be obtained 1.56X 108 (FIG. 1, A), while the conventional microcarrier culture methods, with IL serum-containing cell culture medium can be 3.17 X IO9 cells (FIG. 1, B), while the use of the present invention microcarrier serum-free culture system, IL medium obtained 5.78 X IO9 cells (FIG. 1, C).

[0014] 图2、不同微载体培养下流式细胞仪检测细胞凋亡率的比较:采用传统的微载体培养得到细胞,细胞的凋亡率为15.6%,而在本发明培养系统下细胞的凋亡率仅为5.2%。 [0014] FIG. 2, comparing the apoptosis rate by flow cytometry under various culture microcarriers: traditional microcarrier culture the cells obtained, cells apoptosis rate was 15.6%, while the withered cells in a culturing system of the present invention the death rate was 5.2%. [0015] 图3、保存液保存细胞24小时后细胞凋亡情况检测:运用本发明的细胞保存液,在4度24小时后细胞的状态仍然保持较高的活性。 [0015] FIG 3, Preservation Solution After 24 hours the cells apoptosis detection: Application of cell preservation solution according to the present invention, the state of the cell remains at high activity after 4 degrees for 24 hours. A为系统性红斑狼疮治疗用保存液,B为心脑血管治疗用保存液和C为成骨治疗用保存液。 A systemic lupus erythematosus treated with preservation solution, B is a cardiovascular therapeutic preservation solution C and treated with osteogenic preservation solution.

[0016] 图4、系统性红斑狼疮治疗用保存液保存MSC细胞24h后,MSC细胞炎症因子mRNA水平加强。 [0016] FIG. 4, the treatment of systemic lupus erythematosus save MSC cells after 24h, MSC cytokine mRNA levels reinforcing preservation solution. A为普通林格氏液保存,B为运用本发明专利保存液保存。 A normal Ringer's solution is saved, B is a patent application of the present invention Preservation Solution.

具体实施方式: detailed description:

[0017] 本发明公布了一种优化的脐带来源MSC的规模化培养方式,其优势是在简便、快捷、经济和无外源蛋白,可以直接于临床运用。 [0017] The present invention discloses a way to optimize the large-scale cultivation of umbilical cord-derived MSC, and its advantage is simple, fast, economical, and without exogenous proteins, can be directly in clinical use. 下面就具体实施例来说明本发明的具体运用。 Following specific examples illustrate specific application of the present invention.

[0018] 实例I脐带MSC初步获取: [0018] Examples of umbilical cord MSC preliminary I get:

[0019] 1.脐带选择:选取经过严格的病原体检测(梅毒螺旋体、艾滋病病毒(HIV)、巨细胞病毒(CMV)、乙肝病毒(HBV)、丙肝病毒(HCV)、梅毒、支原体等微生物病原体检测)的产妇,确认安全后使用。 [0019] The umbilical cord selection: Select rigorous pathogen detection (Treponema pallidum, the AIDS virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (of HCV), syphilis, mycoplasma and other microbial pathogens detected ) mothers, confirmed safe to use. 剪取剖宫产产妇手术中近胎盘端脐带20-30cm,置于无菌预冷的PBS中保存。 Cesarean section surgical clip end near the placenta umbilical cord 20-30cm, placed in sterile PBS prechilled saved.

[0020] 2.脐带初步处理:在超净工作台中,将脐带取出放入玻璃培养皿中,剪成约5cm长的小段,用PBS洗净后,剥离脐动脉和脐静脉,并将剥离血管的脐带组织放于DMEM/F12培养基中用手术剪刀剪成2_3左右的组织块。 [0020] 2. umbilical preliminary process: In a laminar flow hood, to remove the umbilical cord into a glass dish, cut into about 5cm-long pieces, after washing with PBS, the umbilical artery and umbilical vein stripping, and the stripping vessel the umbilical cord tissue placed in DMEM / F12 medium with surgical scissors cut about 2_3 tissue mass.

[0021] 3.配置CDH消化酶(II型胶原酶250U/ml,中性蛋白酶100U/ml,透明质酸酶IOU/ml),37度溶解于DMEM/F12培养基,过滤0.22 μ m的滤器除菌备用。 [0021] Configuration CDH digestive enzymes (collagenase type II 250U / ml, neutral protease 100U / ml, hyaluronidase IOU / ml), 37 degrees was dissolved in DMEM / F12 medium, filtration of 0.22 μ m filter alternate sterilization. 消化酶最好现配现用,在4度保存时间勿超过I周。 Preferably digestive enzymes now with the current, at 4 weeks storage time do not exceed I.

[0022] 4.剪碎的组织与⑶H混合酶溶液按1:1的体积比例混合与50ml离心管中,置于37°C摇床中,200rpm振荡,消化3h左右(待组织块基本消化即可,少数较小的组织块的残留并不影响MSC获取效率)。 [0022] 4. The minced tissue with the enzyme solution ⑶H mixed 1: 1 with a volume mixing ratio of 50ml centrifuge tube and placed in 37 ° C shaker, shaken at 200 rpm, about 3H digestion (i.e., the tissue to be digested basic block can be, the smaller the residual tissue mass minority MSC does not affect the extraction efficiency).

[0023] 5.将消化后的组织液4°C,300g离心5min,弃上清,弃上清,将沉淀用PBS重悬于50ml新鲜培养基中,4°C,300g离心5min,弃清,PBS洗两次,注意不要将细胞和组织沉淀弃去。 [0023] 5. The digested tissue was 4 ° C, 300g centrifugation 5min, discarded the supernatant, the supernatant discarded, the pellet resuspended in 50ml PBS with fresh medium, 4 ° C, 300g centrifuge 5min, clear discarded, washed twice with PBS, cells and tissues careful not to precipitate discarded. 最后一遍离心后,弃上清,将沉淀重悬于含有12%胎牛血清的DMEM/F12培养基中,铺入T-25培养瓶。 After the final pass centrifugation, supernatant was discarded and the pellet resuspended in PBS containing 12% fetal calf serum in DMEM / F12 medium, plated in T-25 flasks.

[0024] 6.待细胞长至80%丰度时(约8天),用细胞消化液(0.25%胰酶+0.02^^0丁八),收取细胞。 [0024] 6. When the cells grew to 80% when the abundance (about 8 days), cells with digestion solution (0.25% trypsin + 0.02 0 D ^^ h), cells were harvested.

[0025] 实例2脐带MSC的微载体无血清培养: [0025] Example 2 umbilical cord serum-free culture microcarrier MSC:

[0026] 1、微载体处理:肝素交联葡聚糖微载体在用不含Ca2+和Mg2+离子的PBS中洗涤3次后,在121°C高压灭菌15min,并在4°C下无菌保存。 [0026] 1, microcarrier process: heparin cross-linked dextran microcarriers with free Ca2 + ion and Mg2 + PBS After 3 washes, 15min, and aseptically at 4 ° C for autoclaving at 121 ° C save. 使用前微载体先用培养基洗涤,并在接种前24小时转移到生物反应器Cellbag中进行平衡。 First before using microcarriers washed with medium and transferred into the bioreactor Cellbag 24 hours before inoculation in balance. 为了防止微载体吸附到玻璃表面,所有使用的转移瓶和转瓶都进行硅化处理。 To prevent microcarriers adsorbed to the glass surface, all used transfer bottles and spinner flasks are silicided.

[0027] 2、微载体无血清培养:微载体密度为3g/l,接种密度为0.3X IO6细胞/ml,接种在Cellbag-0.5L 的生物反应器中,以IOrpm, 6°,Ph7.0-7.2,37。 [0027] 2, serum-free culture microcarrier: microcarrier density of 3g / l, seeding density of 0.3X IO6 cells / ml, seeded in Cellbag-0.5L bioreactor in order IOrpm, 6 °, Ph7.0 -7.2,37. . ,5% C02 为培养条件。 , 5% C02 for the culture conditions. 24 小时,细胞贴壁后,旋转速度改为30rpm,7°,其它条件不变,每2天半量换液。 24 hours, the cells adherent to the rotational speed of 30rpm, 7 °, the other conditions remain unchanged, the amount was changed every 2 days and a half.

[0028] 3、细胞收集:每天取样,结晶紫观察,在10天左右,待细胞长满后,停止旋转。 [0028] 3, cells were collected: sampling day, crystal violet was observed at about 10 days, until the cells covered, stop rotating. 长满细胞的微载体沉降后,用PBS收集,洗涤两次后,用0.25%的胰酶消化后收取细胞,并用PBS清洗以备后用。 After the cell covered microcarriers settle, collected in PBS, washed twice with 0.25% trypsin digestion after the cells were harvested and washed with PBS for later use.

[0029] 实例3治疗用MSC保存液运用: [0029] Application Example 3 Treatment with MSC preservation solution:

[0030] 1、三种保存液的配制:严格按照无菌操作,取临床用林格氏液250ml,加入临床用人白蛋白2.5ml和25万AxaIU/ml的临床用肝素纳,混勻后平均分为三份,每份80ml。 In strict accordance with aseptic average, the clinical use of Ringer's solution to take 250ml, was added 2.5ml clinical human albumin and 250,000 AxaIU / ml sodium heparin for clinical use, and after mixing: [0030] 1, three kinds of storage solution formulation It is divided into three, each 80ml. 分别在每份中分别加入:10_7M雌激素和50ng/ml的IL-1 ^ (系统性红斑狼疮治疗用)、50ng/ml的人Midkine (心脑血管治疗用)和100ng/ml的人骨形成蛋白BMP (成骨治疗用)。 Respectively, were added in each portion: human IL-1 ^ (treatment with systemic lupus erythematosus), 50ng / ml of the estrogen and 10_7M of 50ng / ml of Midkine (cardiovascular therapeutic) and 100ng / ml of human bone morphogenetic protein BMP (osteogenic therapeutic).

[0031] 2、分别在每份保存液中加入MSC细胞,细胞浓度为5X IO6个细胞/ml。 [0031] 2, were added to each MSC cells in the preservation solution, the concentration of the cells 5X IO6 cells / ml. 4°C保存24小时后,流式细胞仪检测细胞凋亡情况。 After 24 hours at 4 ° C, flow cytometry apoptosis.

[0032] 3、用Q-PCR方法检测系统性红斑狼疮治疗用MSC保存液保存24h后Cox_2、IL-8、IL-6和CXCR4因子的表达情况。 [0032] 3, the expression Cox_2, IL-8, IL-6 and 24h after storage CXCR4 factor storage solution with MSC detected by Q-PCR treatment of systemic lupus erythematosus.

Claims (4)

1.一种肝素交联葡聚糖微载体,其特征是在传统的CytodeX3微载体制备过程中,增加按照变性胶原/肝素(10: I比例)混合与交联葡聚糖基架化学偶联。 A heparin cross-linked dextran microcarriers wherein the conventional manufacturing process CytodeX3 microcarrier, increased by a denatured collagen / heparin (10: I ratio) was mixed with chemically cross-linked dextran conjugate base frame .
2.一种微载体细胞培养系统,其特征是运用权利要求1所述的微载体,密度为3g/l肝素交联葡聚糖微载体,接种密度为0.3 X IO6细胞/ml,接种在Cellbag-1L的生物反应器中,以IOrpm,6°,Ph7.0-7.2,37°C,5% C02 为培养条件。 A microcarrier cell culture system, wherein said microcarrier use of claim 1, a density of 3g / l heparin cross-linked dextran microcarrier inoculation density of 0.3 X IO6 cells / ml, seeded in Cellbag -1L bioreactor in order IOrpm, 6 °, Ph7.0-7.2,37 ° C, 5% C02 for the culture conditions.
3.—种MSC细胞无血清微载体培养基:其主要成分为AM-V培养基,里面加入人FGF-2 (50ng/ml),人Midkine (60ng/ml),大豆胰酶抑制剂(5ng/ml),青霉素(100units/ml)和链霉素(100units/ml)。 3.- MSC cells serum-free seed microcarrier culture: the main component of medium AM-V, which was added human FGF-2 (50ng / ml), human Midkine (60ng / ml), soybean trypsin inhibitor (5ng / ml), penicillin (100units / ml) and streptomycin (100units / ml).
4.一种系统性红斑狼疮治疗用MSC保存液,其主要成为为:99%临床用林格氏液,1%临床用人白蛋白,1000AxaIU/ml临`床用肝素纳,1(T7雌激素和50ng/ml的IL-1 3。 An MSC systemic lupus erythematosus treated with the preservative solution, which becomes mainly as follows: 99% for clinical use Ringer's solution, 1% human albumin in clinical, 1000AxaIU / ml sodium heparin clinical `bed 1 (the T7 estrogen and 50ng / ml of IL-1 3.
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