CN103583511B - One kind of the mesenchymal stem cell cryopreservation and Injection - Google Patents

One kind of the mesenchymal stem cell cryopreservation and Injection Download PDF

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CN103583511B
CN103583511B CN201310568189.XA CN201310568189A CN103583511B CN 103583511 B CN103583511 B CN 103583511B CN 201310568189 A CN201310568189 A CN 201310568189A CN 103583511 B CN103583511 B CN 103583511B
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mesenchymal stem
injection
present invention
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CN103583511A (en
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任莹莹
田甜
冯学蓉
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四川新生命干细胞科技股份有限公司
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Abstract

本发明公开了一种间充质干细胞冻存液,它包含如下体积百分比的成分:勃脉力A液25~75%、18AA5%复方氨基酸注射液10~35%、20%人血白蛋白1~50%、DMSO5~20%。 The present invention discloses a mesenchymal stem cell freezing medium, which contains the following volume percent composition: A solution Plasmalyte 25 ~ 75%, 18AA5% Amino Acid Injection of Compound 10 to 35%, 20% human serum albumin 1 ~ 50%, DMSO5 ~ 20%. 本发明还提供了一种间充质干细胞注射液及其制备方法。 The present invention also provides a mesenchymal stem cell injection and its preparation method. 本发明间充质干细胞冻存液可克服现有细胞冻存液的缺陷,既能长时间维持间充质干细胞的活率,又能直接临床输注,具有良好的应用前景和经济效益。 Between the present invention mesenchymal stem cells can overcome the disadvantages of the prior cryopreservation cryopreservation of cells, both long time to maintain between mesenchymal stem cells viability, but also direct clinical infusion, has good application prospect and economic benefits.

Description

一种间充质干细胞冻存液及注射液 One kind of the mesenchymal stem cell cryopreservation and Injection

[0001] 本申请是专利申请号:201210321533. 0,申请人:四川新生命干细胞科技股份有限公司,申请日:2012年9月3日的分案申请。 [0001] This application is a patent application number: 2012103215330 applicant: Sichuan New Life Stem Cell Technology Co., filing date: 2012 divisional application is September 3.

技术领域 FIELD

[0002] 本发明涉及一种间充质干细胞冻存液及注射液。 [0002] The present invention relates to a mesenchymal stem cell cryopreservation and injection.

背景技术 Background technique

[0003] 干细胞(stemcells,SC)是一类具有自我复制能力(self-renewing)的多潜能细胞,在一定条件下,它可以分化成多种功能细胞。 [0003] Stem cells (stemcells, SC) is a class of pluripotent cells capable of autonomous replication (self-renewing) is, under certain conditions, it can differentiate into a variety of cellular functions. 干细胞(StemCell)是一种未充分分化, 尚不成熟的细胞,具有再生各种组织器官和人体的潜在功能,医学界称为"万用细胞"。 Stem cells (StemCell) is a not fully differentiated, immature cells that have the regeneration of various tissues and organs of the body's potential function, the medical profession called "universal cell."

[0004] 间充质干细胞【mesenchymal stem cells, MSC】是干细胞家族的重要成员,来源于发育早期的中胚层和外胚层。 [0004] The mesenchymal stem cells [mesenchymal stem cells, MSC] Stem cells are an important member of the family, from the development of early mesoderm and ectoderm. MSC最初在骨髓中发现,因其具有多向分化潜能、造血支持和促进干细胞植入、免疫调控和自我复制等特点而日益受到人们的关注。 MSC was originally found in the bone marrow, because of pluripotent hematopoietic stem cell engraftment support and promote immune regulation and self-replication features and increasing people's attention. 间充质干细胞在体内或体外特定的诱导条件下,可分化为脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞,连续传代培养和冷冻保存后仍具有多向分化潜能,可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。 After the mesenchymal stem cells in vivo or in vitro specific induction conditions, differentiate into fat, bone, cartilage, muscle, tendons, ligaments, nerves, liver, cardiac muscle, endothelial and other cells, subcultured and cryopreservation still with multiple differentiation potential, it can be used as an ideal seed cells for tissue and organ repair damage caused by aging and disease.

[0005] 由MSCs制备的干细胞注射液在临床上可以用于预防和治疗异体造血干细胞移植后产生的移植物抗宿主病(GVHD),以及治疗自生免疫性疾病,I型糖尿病、如红斑狼疮、风湿性关节炎和硬皮病等。 [0005] Stem cell injection prepared from MSCs clinically useful for preventing and treating allogeneic hematopoietic stem cells transplantation graft-versus-host disease (of GVHD), as well as therapeutic autogenous autoimmune diseases, type I diabetes, such as lupus erythematosus, rheumatoid arthritis and scleroderma. 然而,与普通生物制品和药品不同,间充质干细胞注射液的活性物质是细胞,细胞在离体环境中,活性会迅速下降,传统方法用含人血白蛋白的氯化钠注射对MSCs进行保存,在8~24小时以内,细胞活性即大幅下降,无法长期保证细胞活性,给MSCs 注射液大量、全国大范围临床应用于提出了严峻的挑战。 However, biological products and pharmaceuticals with different common mesenchymal stem cell injection the substance is cells, in vitro environment, the activity will decline rapidly, the conventional method and sodium chloride injection containing human serum albumin to be MSCs save, within 8 to 24 hours, cell activity that is dropped, we can not guarantee long-term viability, to a large number of MSCs injection, used in a wide range of national clinical poses a severe challenge.

[0006] 大量研宄发现,用含有胎牛血清或者细胞培养基的保存液可以解决细胞活性下降快的问题,能够长时间维持细胞活率。 [0006] large study based found that a preservation solution containing fetal bovine serum or cell culture media can solve the problem of fast cytotoxicity, cell viability can be maintained for a long time. 但是,细胞接触胎牛血清时会内吞胎牛血清,进而引起细胞某些蛋白表达特性的变化,细胞回输人体后可能引起过敏反应,不能直接用于临床输注;细胞培养基因其成分与人体环境相差较远,未被批准临床使用,也不能直接临床输注,通常被科研机构用来体外培养及冻存细胞。 However, when the cells would contact the inner swallow FBS fetal bovine serum, thereby causing a change in expression of certain proteins characteristic of the cell, cell reinfusion human body may cause allergic reactions, can not be directly used for clinical infusion; cell culture medium and its constituents human environment are far away, has not been approved for clinical use, can not be directly infused clinical, research institutions are usually used in vitro cell culture and cryopreservation.

[0007] 申请号:201010246409. 3,发明名称:一种直接静脉应用的间充质干细胞冻存液的专利申请,公开了一种间充质干细胞冻存液的制备方法,包括如下步骤:1)将人AB血置于无菌离心管中,在1000rpm-1200rpm条件下离心15分钟;2)吸取上清液,转移入新的离心管中,在4000g条件下离心20分钟,上清即为冻存用人AB血浆;3)将二甲基亚砜、上述冻存用人AB血浆、勃脉力A注射液按体积比1 : 3 : 6混合均匀,即为间充质干细胞冻存液。 [0007] application number: 2010102464093, entitled: intravenous application between a direct Patent Application mesenchymal stem cell cryopreservation is disclosed a method for preparing inter mesenchymal stem cell cryopreservation, comprising the following steps: ) human AB serum placed in a sterile centrifuge tube at 1000rpm-1200rpm centrifuged 15 minutes; 2) the supernatant transferred into a new centrifuge tube, centrifuged at 4000g for 20 minutes, the supernatant namely frozen human AB plasma; 3) dimethylsulfoxide the frozen human AB plasma, Plasmalyte A injection volume ratio of 1: 3: 6 mixed, that is, between the mesenchymal stem cell cryopreservation. 由该专利申请文件的实施例表1可以看出,该发明的冻存液I和II冻存1、2、3个月后复苏, 检测细胞活率,冻存液I的细胞活率依次为96%、92. 6%和89%,其细胞活率降低率为3. 5%/ 月,冻存液II细胞活率依次为97. 4%、95%和为94. 7%,其细胞活率降低率为1. 35%/月,该申请公开的冻存液虽然可以直接静脉输注,但是细胞活率维持时间短。 It can be seen from Example 1 of this patent application document table, cryopreservation solution I and II of the invention are 2,3 months after cryopreservation recovery, detecting cell viability, cell viability were frozen in liquid I 96%, 92.6% and 89%, which reduced cell viability was 3.5% / month, cell viability cryopreservation solution II were 97.4%, 95%, and was 94.7%, the cell reducing the survival rate was 1.35% / month, the disclosure of which cryopreservation solution, although direct intravenous infusion, but the cell viability short duration. 并且,该细胞冻存液中含有人AB血浆,血浆中含有大量未知成分,用于异体输注存在安全隐患,同时,血浆来源也很少,成本高,难以大规模应用。 Further, the cryopreservation liquid containing human AB plasma, plasma contains a large number of unknown components, for infusions allogeneic unsafe, while rarely plasma source, high cost, large-scale application difficult.

[0008] 综上,现有技术中的细胞的冻存液难以兼顾长时间维持细胞活率与临床直接输注。 [0008] In summary, the cells were frozen prior art is difficult to achieve long time to maintain cell viability and clinical direct infusion.

发明内容 SUMMARY

[0009] 为了解决上述问题,本发明提供了一种新的间充质干细胞冻存液。 [0009] In order to solve the above problems, the present invention provides a new mesenchymal stem cell cryopreservation.

[0010] 本发明间充质干细胞冻存液,它包含如下体积百分比的成分: [0010] The present invention is between mesenchymal stem cell freezing medium, comprising the following percentage composition by volume:

[0011] [0011]

Figure CN103583511BD00041

[0012] 其中,上述冻存液的pH为6. 0~6. 8,渗透压为300~540m0smol/Kg。 [0012] wherein, pH of the above solution is frozen 6.0 ~ 6.8, osmotic pressure of 300 ~ 540m0smol / Kg.

[0013] 优选地,所述冻存液包含如下体积百分比的成分: [0013] Preferably, the frozen liquid composition comprising the following percentages by volume:

[0014] [0014]

Figure CN103583511BD00042

[0015] 进一步优选地,所述冻存液包含如下体积百分比的成分: [0015] Further preferably, the frozen solution containing the following percentages by volume of the composition:

[0016] 它包含如下体积百分比的成分: [0016] It contains the following percentages by volume of the composition:

[0017] [0017]

Figure CN103583511BD00043

[0018] 进一步优选地,所述冻存液由如下体积百分比的成分组成: [0018] Further preferably, the frozen liquid volume percentage by the following chemical composition:

[0019] [0019]

Figure CN103583511BD00044

[0021] 勃脉力A液:每1000 ml含氯化钠5. 26g、葡萄糖酸钠5. 02g、醋酸钠3. 68g、氯化钾〇• 37g、氯化镁0• 30g; [0021] Plasmalyte A solution: sodium chloride containing per 1000 ml 5. 26g, sodium gluconate 5. 02g, sodium acetate 3. 68g, potassium square • 37g, magnesium 0 • 30g;

[0022] 20%人血白蛋白:是药典规定的人血白蛋白制品,其中,人血白蛋白的浓度为20% (v/v)〇 [0022] 20% human serum albumin: human serum albumin products Pharmacopoeia, wherein the concentration of human serum albumin to 20% (v / v) square

[0023] 18AA5%复方氨基酸注射液:由18种氨基酸与山梨醇配制而成的灭菌水溶液。 [0023] 18AA5% compound amino acid injection: prepared from 18 kinds of amino acids with sorbitol made sterile aqueous solutions. 每1000 ml含:L-脯氨酸I. 00g、L-丝氨酸I. 00g、L-丙氨酸2. 00g、L-异亮氨酸3. 52g、L-亮氨酸4. 90g、L-门冬氨酸2. 50g、L-酪氨酸0. 25g、L-谷氨酸0. 75g、L-苯丙氨酸5. 33g、L-精氨酸盐酸盐5. 00g、L-赖氨酸盐酸盐4. 30g、L-缴氨酸3. 60g、L-苏氨酸2. 50g、L-组氨酸盐酸盐 Each 1000 ml containing: L- proline I. 00g, L- serine I. 00g, L- alanine 2. 00g, L- isoleucine 3. 52g, L- leucine 4. 90g, L - aspartate 2. 50g, L- tyrosine 0. 25g, L- glutamic 0. 75g, L- phenylalanine 5. 33g, L- arginine hydrochloride 5. 00g, L - lysine 4. 30g, L- histidine pay 3. 60g, L- threonine 2. 50g, L- histidine hydrochloride

[0024] (C6H9N302 •HCl•H20) 2. 50g、L-色氨酸0•90g、L-甲硫氨酸2. 25g、L-胱氨酸0• 10g、甘氨酸7. 60g、山梨醇50. 00g、亚硫酸氢钠0• 5g。 [0024] (C6H9N302 • HCl • H20) 2. 50g, L- tryptophan 0 • 90g, L- methionine 2. 25g, L- cystine 0 • 10g, glycine 7. 60g, sorbitol 50 . 00g, sodium bisulfite 0 • 5g.

[0025] DMSO :二甲基亚砜。 [0025] DMSO: dimethyl sulfoxide.

[0026] 本发明还提供了一种间充质干细胞注射液,其包含前述间充质干细胞冻存液和间充质干细胞,间充质干细胞的浓度为IXIO5~IX108个/ml。 [0026] The present invention also provides a mesenchymal stem cell injection, comprising between the mesenchymal stem cell cryopreservation and mesenchymal stem cells, mesenchymal stem cells at a concentration of IXIO5 ~ IX108 cells / ml. 间充质干细胞的浓度根据临床需求调整。 Mesenchymal stem cell concentration adjusted according to clinical needs.

[0027] 本发明还提供一种制备前述间充质干细胞注射液的方法,它包括如下步骤: [0027] The present invention also provides a method for preparing the inter mesenchymal stem cell injection, comprising the steps of:

[0028] (1)按照前述比例取勃脉力A液、18AA5%复方氨基酸注射液、20%人血白蛋白和DMSO; [0028] (1) in accordance with the proportion of the liquid taken Plasmalyte A, 18AA5% compound amino acid injection, 20% human serum albumin and DMSO;

[0029] (2)将步骤(1)勃脉力A液、18AA5%复方氨基酸注射液和20%人血白蛋白混勾,加入间充质干细胞至细胞浓度为IXIO5~IX10 8个/ml,得细胞悬液; [0029] (2) The step (1) Plasmalyte A solution, 18AA5% compound amino acid injection and 20% human serum albumin mix hook, between the addition of mesenchymal stem cells to a cell concentration of IXIO5 ~ IX10 8 / ml, and have cell suspension;

[0030] (3)将步骤(I)DMSO加入步骤(2)所得的细胞悬液,即可。 [0030] (3) the step (I) DMSO cell suspension was added in step (2) obtained can.

[0031] 制备得到的间充质干细胞注射液,程控降温后保存在液氮罐里,使用时,取出复苏即可。 Between [0031] prepared mesenchymal stem cell injection, programmed freezing in liquid nitrogen and stored jar, in use, to remove recovery.

[0032] 本发明间充质干细胞冻存液,可以长时间维持间充质干细胞的活性,不含细胞培养基、胎牛血清、人血浆等不安全成分,制备的间充质干细胞注射液,冻存复苏后可直接用于临床输注,安全有效,制备方法简单,成本低廉,具有良好的临床应用前景。 [0032] The present invention is between mesenchymal stem cell freezing medium, long periods of activity of mesenchymal stem cells, cell-free culture medium, fetal bovine serum, human plasma and other components between unsafe maintained between mesenchymal stem cells prepared injection, after cryopreservation can be directly used for clinical transfusion, safe and effective, simple preparation method, low cost and has good prospects for clinical application.

[0033] 显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。 [0033] Obviously, the above-described present invention, according to the conventional technical knowledge and customary practice in the art, without departing from the present invention, the above-described basic technical idea premise, may be made of other forms of modification, replacement or change.

[0034] 以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。 [0034] DETAILED DESCRIPTION The following embodiments by the form of the above-described present invention will be further described in detail again. 但不应将此理解为本发明上述主题的范围仅限于以下的实例。 However, this should not be understood that the scope of the present invention, the above subject is limited to the following examples. 凡基于本发明上述内容所实现的技术均属于本发明的范围。 Where the above-described technique based on the present invention are achieved within the scope of the present invention.

附图说明 BRIEF DESCRIPTION

[0035] 图1生长曲线对比图 [0035] FIG 1 FIG growth curve comparison

[0036] 图2细胞冻存前生长形态 [0036] FIG 2 before cryopreservation growth morphology

[0037] 图3本发明间充质干细胞注射液冻存1年后复苏后细胞生长形态 [0037] FIG. 3 between the present invention mesenchymal stem cell growth morphology of cryopreserved cell injection after 1 year after resuscitation

[0038] 图4对照组冻存一年后复苏期细胞生长形态 [0038] FIG. 4 controls the recovery of cells after cryopreservation year growth morphology

[0039] 图5本发明间充质干细胞冻存1年复苏后细胞成脂分化能力图[0040]图6未冻存新鲜细胞成脂分化能力图 [0039] mesenchymal stem cell cryopreservation of the present invention between 5 adipogenic differentiation of FIG. 1 after resuscitation [0040] FIG. 6 is not fresh cell adipogenic differentiation FIG frozen

[0041] 图7对照组冻存细胞成脂分化能力图 [0041] FIG. 7 controls frozen cells adipogenic differentiation FIG.

[0042]图8本发明间充质干细胞注射液冻存1年复苏后细胞成骨分化能力图 [0042] FIG. 8 between the present invention mesenchymal stem cell injection frozen cells osteogenic differentiation of FIG. 1 after resuscitation

[0043] 图9未冻存新鲜细胞成骨分化能力图 [0043] FIG. 9 fresh osteogenic differentiation is not frozen FIG.

[0044] 图10对照组冻存细胞成骨分化能力图 [0044] FIG 10 controls osteogenic differentiation frozen FIG

[0045] 图11本发明间充质干细胞注射液冻存1年复苏后48小时后细胞的成脂分化能力图 [0045] FIG. 11 between the present invention mesenchymal stem cell injection cryopreservation FIG adipogenic differentiation ability after 48 hours of cell recovery after 1 year

[0046] 图12本发明间充质干细胞注射液冻存1年复苏后48小时后细胞的成骨分化能力图 [0046] FIG. 12 between the present invention mesenchymal stem cells into osteoblasts injection cryopreservation FIG ability after 48 hours of cell recovery after 1 year

[0047] 图13本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD45检测结果 [0047] FIG. 13 between the present invention mesenchymal stem cell injection in a frozen, cell surface CD45 detection result resuscitation

[0048] 图14本发明间充质干细胞注射液冻存1年,复苏后细胞表面HLA-DR检测结果 [0048] FIG. 14 between the present invention mesenchymal stem cell injection in a frozen, HLA-DR cell surface detection result resuscitation

[0049] 图15本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD14检测结果[0050] 图16本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD34检测结果 Between [0049] 15 of the present invention mesenchymal stem cell injection cryopreservation year, cell surface CD14 detection result resuscitation [0050] Room 16 of the present invention mesenchymal stem cell injection cryopreservation 1 year surface CD34 cell detection result resuscitation

[0051] 图17本发明间充质干细胞注射液冻存1年,复苏后细胞表面79a检测结果 [0051] FIG. 17 between the present invention mesenchymal stem cell injection in a frozen, cell surface 79a detection result resuscitation

[0052] 图18本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD90检测结果 [0052] FIG. 18 between the injection of the present invention mesenchymal stem cells in a cryopreservation, CD90 cell surface detection result resuscitation

[0053] 图19本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD105检测结果 [0053] FIG. 19 between the present invention mesenchymal stem cell injection in a frozen, cell surface CD105 detection result resuscitation

[0054] 图20本发明间充质干细胞注射液冻存1年,复苏后细胞表面CD166检测结果 Between [0054] 20 of the present invention mesenchymal stem cell injection in a frozen, cell surface CD166 detection result resuscitation

[0055] 图21本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD45检测结果 [0055] FIG. 21 between the present invention mesenchymal stem cells in a frozen injection, the detection result of the surface of CD45 cells after 48h recovery

[0056] 图22本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面HLA-DR检测结果 [0056] Figure 22 between the present invention mesenchymal stem cells in a frozen injection, the detection result of surface HLA-DR cells 48h after resuscitation

[0057] 图23本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD14检测结果 [0057] FIG. 23 between the injection of the present invention mesenchymal stem cells cryopreserved for 1 year, the results of the detection of CD14 cells after 48h recovery

[0058] 图24本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD34检测结果 [0058] FIG. 24 between the injection of the present invention mesenchymal stem cells in a cryopreservation, the detection result of surface CD34 cells 48h after resuscitation

[0059] 图25本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面79a检测结果 [0059] FIG. 25 between the present invention mesenchymal stem cells in a frozen injection, 48h recovery cell surface 79a after the detection result

[0060] 图26本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD90检测结果 Between [0060] 26 of the present invention mesenchymal stem cells in a frozen injection, 48h CD90 cell surface detection result resuscitation

[0061] 图27本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD105检测结果 [0061] FIG. 27 between the present invention mesenchymal stem cells in a frozen injection, the detection result of surface CD105 cells 48h resuscitation

[0062] 图28本发明间充质干细胞注射液冻存1年,复苏后48h细胞表面CD166检测结果 [0062] FIG. 28 between the injection of the present invention mesenchymal stem cells in a cryopreservation, 48h of cell surface CD166 detection result resuscitation

具体实施方式 Detailed ways

[0063] 主要仪器及试剂: [0063] The main instruments and reagents:

[0064] 仪器:离心机Eppendorf5810R、流式细胞仪BeckmanFC500、细胞计数仪罗氏CASY Model77、倒置相差显微镜奥林巴斯CKX41、C02培养箱Thermo8000; [0064] Instrument: Centrifuge Eppendorf5810R, flow cytometry BeckmanFC500, cell counter Roche CASY Model77, Olympus inverted microscope CKX41, C02 incubator Thermo8000;

[0065] 试剂: [0065] Reagents:

[0066] 0. 9%氯化钠注射液购自四川科伦药业股份有限公司生产 [0066] 0.9% Sodium Chloride Injection, available from four coron Pharmaceutical Co.

[0067]18AA5%氨基酸复方注射液购自山东鲁抗辰欣药业有限公司 [0067] 18AA5% amino acid compound injection available from Shandong Lukang Pharmaceutical Co., Ltd. Chenxin

[0068] 勃脉力A液(PLASMA-LYTE)购自上海百特医疗用品有限公司 [0068] Plasmalyte A liquid (PLASMA-LYTE) were purchased from Shanghai Baxter Healthcare Ltd.

[0069]20%人血白蛋白购自成都蓉生药业有限责任公司 [0069] 20% human serum albumin were purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0070] DMSO购自WAK-Chemie [0070] DMSO was purchased from WAK-Chemie

[0071] LG-DMEM/F12基础培养基+10%胎牛血清购自GIBCO [0071] LG-DMEM / F12 base medium + 10% fetal bovine serum were purchased from GIBCO

[0072] 人间充质干细胞无血清培养基购自GIBCO [0072] The human mesenchymal stem cells were purchased from GIBCO serum-free medium

[0073] 0• 05% 胰酶-0• 0296EDTA溶液购自GIBCO [0073] 0 • 05% -0 • 0296EDTA trypsin was purchased from GIBCO

[0074]0• 25%胰酶-0• 38g/L EDTA溶液购自GIBCO [0074] 0 • 25% pancreatin -0 • 38g / L EDTA solution was purchased from GIBCO

[0075] 0. 04%台盼蓝染色液购自GIBCO [0075] 0.04% trypan blue staining solution purchased from GIBCO

[0076] 人间充质干细胞成骨分化试剂盒购自GIBCO [0076] The human mesenchymal stem cells into osteoblasts kit purchased from GIBCO

[0077] 人间充质干细胞成脂分化试剂盒购自GIBCO [0077] The human mesenchymal stem cells in adipogenic differentiation kit purchased from GIBCO

[0078] 茜素红S购自Sigam [0078] Alizarin Red S were purchased from Sigam

[0079] 油红0购自Sigam [0079] Oil Red O available from Sigam

[0080] 多聚甲醛购自Sigam [0080] available from paraformaldehyde Sigam

[0081] 实施例1用本发明间充质干细胞冻存液制备注射液 [0081] Example 1 of the present invention is between mesenchymal stem cell cryopreservation preparation of injectables

[0082] 1、制备本发明间充质干细胞注射液 [0082] 1, the present invention is prepared between mesenchymal stem cell injection

[0083] 如表1所示冻存液的配方,取勃脉力A液、18AA5%复方氨基酸注射液和20%人血白蛋白,混匀,加入脐带间充质干细胞至细胞密度为IXIO7个/ml,再加入DMSO,即可。 [0083] As shown in Table 1 Formulation cryopreservation, take Plasmalyte A solution, 18AA5% compound amino acid injection and 20% human serum albumin, mixing, addition of umbilical cord mesenchymal stem cells to a cell density of IXIO7 / ml, was added DMSO, to.

[0084] 表1本发明间充质干细胞冻存液配方 [0084] Table 1 Invention between mesenchymal stem cell cryopreservation formula

[0085] [0085]

Figure CN103583511BD00071

[0086] 组6中,白蛋白含量高,成本较其他组高。 In [0086] Group 6, high albumin content, higher cost than other groups.

[0087] 对照组:取90ml完全培养基(90% (v/v)DMEM/F12+10% (v/v)胎牛血清),混匀,加入脐带间充质干细胞至细胞密度为IXIO7个/ml,再加入10mlDMSO,即可。 [0087] Control group: 90ml complete medium (90% (v / v) DMEM / F12 + 10% (v / v) fetal calf serum), mix, add umbilical cord mesenchymal stem cells to a cell density of IXIO7 / ml, was added 10mlDMSO, can. 对照组的配方是国内作为常用的细胞冻存液配方。 It is the formulation of the control group as commonly used in cryopreservation fluid formulations.

[0088] 2、细胞活率检测 [0088] 2, cell viability detection

[0089] 取冻存前细胞,测试细胞活率;将本发明制备的间充质干细胞注射液以及对照组, 程控降温后保存在液氮罐里,冻存1年后在37°C水浴复苏,测试细胞活率。 [0089] before removing the frozen cells, cell viability test; the present invention is prepared between MSCs after injection and the control group, stored in liquid nitrogen cooling pot programmed, frozen in a water bath at 37 ° C after recovery test cell viability.

[0090] 检测方法为台盼蓝经典染色法。 [0090] Detection of Classical trypan blue staining.

[0091] 3、检测结果 [0091] 3, the detection result

[0092] 结果如表2所示: [0092] The results are shown in Table 2:

[0093] 表2细胞活率实验结果 [0093] TABLE 2 cell viability experiments

Figure CN103583511BD00072

Figure CN103583511BD00081

[0095] 由表2可以看出: [0095] As can be seen from Table 2:

[0096] 本发明间充质干细胞冻存液制备的注射液,冻存一年以后复苏,细胞活率均在89. 2%之上,与冻存前相比,细胞活率最多仅下降了7. 6%/年,经计算,细胞活率降低率最多仅为0. 63%/月,下降幅度极低;在本发明优选范围内,可以进一步提高细胞活率,降低生产成本,同时,注射液的pH、渗透压接近人体的pH、渗透压,更为安全;其中,组3的细胞活率最高,为95. 4%,pH为6. 6~6. 8,渗透压为320~360m0smol/Kg,最接近人体pH和渗透压,安全有效,为最佳配比。 [0096] Injection preparation of mesenchymal stem cell cryopreservation between the present invention, cryopreservation recovery year later, cell viability rate over 89.2%, compared with that before freezing, cell viability decreased only up 7.6% / year, is calculated, cell viability was only decreased the rate of up to 0.63% / month, the decline is very low; within the preferred range of the present invention can be further improved cell viability, reduce production costs, at the same time, injection pH, osmolarity of the human body near pH, osmotic pressure, safer; wherein the cell viability of the highest group 3, was 95. 4%, pH of 6.6 ~ 68, 320 ~ osmotic pressure. 360m0smol / Kg, the closest body pH and osmolality, safe and effective, the best ratio.

[0097] 实验说明,本发明间充质干细胞冻存液可以长时间维持细胞活率。 [0097] The experiments described, the present invention is between mesenchymal stem cell cryopreservation cell viability can be maintained for a long time.

[0098] 根据本实施例结果,选取本发明最佳配比的间充质干细胞冻存液做进一步研宄: [0098] According to the present embodiment As a result, the present invention is to select the best ratio mesenchymal stem cell cryopreservation for further study based on:

[0099] 实施例2本发明间充质干细胞注射液冻存1年后各性能检测[0100] 1、实验方法 [0099] Example embodiments of the present invention between two mesenchymal stem cell injection one year after each performance test [0100] 1, Experimental Methods Cryopreservation

[0101] 本发明间充质干细胞注射液:取与实施例1组3的制备方法相同的另一批次细胞, 冻存1年后在37 °C水浴复苏,保存在2~8度冰箱。 [0101] between the present invention mesenchymal stem cells injection: Group 1 Example take another batch of the same preparation method 3 cells after cryopreservation in a water bath at 37 ° C the recovery is stored in the refrigerator 2 to 8 degrees.

[0102] 对照组:同实施例1。 [0102] Control group: same as in Example 1.

[0103] (1)冻存1年后复苏,复苏后不同时间点的细胞活率检测 [0103] (1) 1 year after frozen recovery after the recovery cell viability at different time points detected

[0104] 取冻存前细胞测试细胞活率;本发明间充质干细胞注射液和对照组复苏后0小时,24小时、48小时和72小时的细胞分别测试细胞活率。 [0104] before removing the frozen cells cell viability test; charge between the present invention mesenchymal stem cells 0 hours after injection and recovery in the control group, 24 hours, 48 ​​hours and 72 hours the cells were cell viability test. 检测方法为台盼蓝经典染色法。 Detection method for the classic trypan blue staining.

[0105] (2)生长曲线对比 [0105] (2) Growth curve of Comparative

[0106] 取冻存前细胞,本发明间充质干细胞注射液和对照组复苏后0小时细胞,用台盼蓝经典染色法计数,分别将以2000个/cm2密度接种在T25培养瓶中,每个组别接种21瓶, 分别每天每个组别计数三个培养瓶中细胞数量,取其平均值,进行生长曲线的绘制。 [0106] Cells taken before freezing, 0 hours cells were counted using trypan blue staining classical mesenchymal stem cell injection and recovery between the control group of the present invention, respectively, will be 2,000 / cm2 in T25 flasks seeded at a density, each group was inoculated bottles 21, each group were counted each day draws three culture flasks number of cells, the mean value, for a growth curve. 没有计数的培养瓶每3天更换一次培养基。 Not counting flasks changing the medium every 3 days.

[0107] (3)细胞生长形态 [0107] (3) cell growth morphology

[0108] 取步骤(2)生长到第5天的细胞进行显微镜下观察不同组别细胞生长的异同,并拍照。 [0108] from step (2) is grown to day 5 cells were similarities and differences between different groups of cells grown under the microscope, and photographed.

[0109] (4)细胞分化能力测试 [0109] (4) cell differentiation ability test

[0110] 取冻存前细胞,本发明间充质干细胞注射液和对照组复苏后〇小时,和48小时的细胞,分别以5000个/cm2的密度接种到6孔板中,待长至70%汇合度时,在培养基中加入分化GIBCO成骨、成脂诱导培养基,每4天更换一次培养基,14天时分别用油红O染色对成脂分化细胞染色,大于21天用茜素红S对成骨分化细胞进行染色。 [0110] before removing the frozen cells, mesenchymal stem square hours after cell injection and recovery between the control group of the present invention, and 48 hours the cells were at a density of 5000 / cm2 were seeded into 6-well plates, grown to be 70 when% confluence, the medium was added in GIBCO osteogenic differentiation, adipogenic medium, changing the medium every 4 days, 14 days, respectively, the cells were stained with oil red O staining for adipogenic differentiation, greater than 21 days with alizarin osteogenic differentiation of red S cells were stained.

[0111] (5)表面标志检测 [0111] (5) detection of surface markers

[0112] 取冻存前细胞,本发明间充质干细胞注射液和对照组复苏后0小时,和48小时的细胞,以FITC标记的CD90、CD45、HLA-DR以及以PE标记的CD105、CD14、CD34、CD79a、CD166 进行流式测定。 [0112] before removing the frozen cells 0 hours after MSCs injection and control groups resuscitation room of the present invention, and 48 hours the cells to FITC-labeled CD90, CD45, HLA-DR and with PE-labeled CD105, CD14 , CD34, CD79a, CD166 assay streaming.

[0113] 2、实验结果 [0113] 2. Experimental results

[0114] (1)细胞活率结果如表3所示: [0114] (1) cell viability The results are shown in Table 3:

[0115] 表3细胞活率实验结果 [0115] Table 3 cell viability experiments

Figure CN103583511BD00091

[0117] 注:国家标准规定,间充质干细胞注射液中的细胞活率不得低于85%。 [0117] Note: national standards, mesenchymal stem cells cell viability injection shall not be less than 85%.

[0118] 由表3可以看出,与冻存前相比,本发明间充质干细胞注射液复苏后0h,细胞活率下降很微小。 [0118] As can be seen from Table 3, compared with that before freezing, the present invention is filled between the stromal cells in the injection recovery 0h, cell viability decreased very small.

[0119] 在冻存复苏后〇h、24h、48h和72h,本发明间充质干细胞注射液的细胞活率均高于对照组的细胞活率,随着时间增加,二者的差距增大,复苏后24h时,前者就显著高于后者了;复苏后,本发明间充质干细胞注射液中细胞活率的下降幅度小,而对照组细胞活率的下降幅度大,前者显著低于后者;本发明间充质干细胞注射液复苏后72h的细胞活率仍然维持在85%以上,复苏后的有效期长达3天,大大方便临床使用,而对照组在复苏后24h,其细胞活率就降到85%左右,复苏后的有效期仅为1天。 [0119] After cryopreservation 〇h, 24h, 48h and 72h, cell viability was clearly higher cell viability between the injection of the present invention mesenchymal stem cells, with increasing time, increases the gap between the two after resuscitation 24h, the former is markedly higher than the latter; the post-resuscitation, between the present invention mesenchymal stem cells small decrease cell viability of injection, the decline in the control group survival rate of cells large, the former is significantly lower than the latter; between the present invention mesenchymal stem cell injection after 72h recovery cell viability remained at 85% or more, after the recovery period up to 3 days, greatly facilitate the clinical use, while the control group 24h, after which cell viability recovery rate dropped to about 85%, the recovery period after only one day.

[0120] (2)生长曲线绘制 [0120] (2) Growth curve drawing

[0121] 生长曲线密度接种约为2000个/cm2,每组每天计三个样取其平均值,结果如表4 和图1所示: [0121] Growth curves seeded at a density of about 2000 / cm2, each set of three samples per day count average value, the results shown in Table 4 and Figure 1:

[0122] 表4生长曲线数据统计 [0122] Table 4 Growth curve statistics

Figure CN103583511BD00092

[0124] 由表4和图1可以看出,与冻存前和对照组相比,本发明间充质干细胞注射液在冻存1年后复苏,增殖能力没有明显变化。 [0124] As can be seen from Table 4 and Figure 1, compared with cryopreserved controls and, between the present invention mesenchymal stem cell recovery after cryopreservation injection one year, no significant changes in proliferation.

[0125] (3)细胞形态对比 [0125] (3) Comparative cell morphology

[0126] 如图2~4所示,本发明间充质干细胞注射液在冻存1年后复苏,细胞形态与冻存前以及对照组细胞的形态没有明显变化,均呈梭形,折光度高,分布均一。 [0126] 2 to 4, between the present invention mesenchymal stem cell recovery after cryopreservation injection one year, before cryopreservation and cell morphology and cell morphology in the control group did not change significantly, showed spindle, diopter high, uniform distribution.

[0127] (4)细胞分化能力测试 [0127] (4) cell differentiation ability test

[0128] 如图5~12所示,本发明间充质干细胞注射液在冻存1年后复苏,其成脂、骨分化能力与冻存前以及对照组细胞的形态没有明显变化,均出现大量的脂肪滴和钙堆积。 [0128] As shown in FIG. 5 to 12, between the present invention mesenchymal stem cell recovery after cryopreservation injection 1, its adipogenic, osteogenic differentiation capability before and morphology of cryopreserved control cells did not change significantly, both appear a lot of fat droplets and calcium accumulation. 复苏后48h,本发明间充质干细胞注射液仍然具有成脂、骨分化能力。 48h, the present invention between the charge recovery after stem cell injection still adipogenic, osteogenic differentiation ability.

[0129] (5)表面标志检测 [0129] (5) detection of surface markers

[0130] 如图13~20所示,本发明间充质干细胞注射液冻存1年后复苏后Oh细胞高表达CD90、CD105、CD166,而不表达CD14、CD34、CD79a、CD45、HLA-DR,符合MSCs表面标志的特征, 说明冻存1年后的细胞仍然为间充质干细胞。 [0130] As shown in FIG. 13 to 20, after the present invention is between mesenchymal stem cells in a cryopreservation recovery injection Oh cells expression CD90, CD105, CD166, and do not express CD14, CD34, CD79a, CD45, HLA-DR , in line with characteristic surface markers of MSCs, indicating frozen cells after 1 year still mesenchymal stem cells.

[0131] 如图21~28所示,本发明间充质干细胞注射液冻存1年后复苏,复苏后48小时后的细胞高表达CD90、CD105、CD166,而不表达CD14、CD34、CD79a、CD45、HLA-DR,符合MSCs 表面标志的特征,说明冻存1年后的细胞仍然为间充质干细胞。 [0131] As shown in FIG. 21 to 28, between the present invention mesenchymal stem cell injection one year after cryopreservation recovery, high expressing cells after 48 hours after resuscitation CD90, CD105, CD166, and do not express CD14, CD34, CD79a, CD45, HLA-DR, characterized in compliance MSCs surface markers, described in 1 cells after cryopreservation still mesenchymal stem cells.

[0132] 实验说明,用本发明冻存液冻存间充质干细胞,细胞的增殖、分化能力均未受到影响,细胞本身也未发生变异,说明本发明冻存液不会影响间充质干细胞的性质;本发明间充质干细胞冻存液,无论是在冻存过程中,还是冻存复苏后,都可以长时间维持细胞活率,制备的注射液有效期长,复苏后有足够的时间给药,临床应用非常方便。 [0132] Experiments described, with the present invention, cryopreservation solution of cryopreserved mesenchymal stem cells, proliferation, differentiation were not affected, nor cells themselves mutate, cryopreservation solution described does not interfere with the present invention, mesenchymal stem cells nature; between the present invention mesenchymal stem cell freezing medium, either in the freezing process, or after cryopreservation, can maintain cell viability for a long time, the period of validity of the injection preparation, there is enough time to recover after medicine, clinical application is very convenient.

[0133] 实施例3本发明间充质干细胞注射液细胞活率与冻存时间的关系 Example 3 The relationship between the present invention mesenchymal stem cell injection and kept frozen cell viability of [0133] Embodiment

[0134] 1、实验方法 [0134] 1, Experimental Methods

[0135] 本发明间充质干细胞注射液:按照实施例1组3配比,制备间充质干细胞注射液, 程控降温后保存在液氮罐里。 [0135] between the present invention mesenchymal stem cells injection: group 1 according to the Example 3 ratio between injection preparation of mesenchymal stem cells, stored in liquid nitrogen cooling programmed jar.

[0136] 对照:同实施例1。 [0136] Control: the same as in Example 1.

[0137] 冻存1、6和12个月后在37°C水浴复苏,分别测试细胞活率,检测方法为台盼蓝经典染色法。 [0137] After freezing, 6 and 12 months of recovery at 37 ° C water bath, cell viability were tested, detection of classical trypan blue staining.

[0138] 2、实验结果 [0138] 2. Experimental results

[0139] 实验结果如表5所示: [0139] The results shown in Table 5:

[0140] 表5细胞活率与冻存时间的关系 [0140] Table 5 Relationship between cell survival rate of cryopreserved time

Figure CN103583511BD00101

[0142] 由表5可以看出,冻存1、6、12个月后复苏,本发明间充质干细胞注射液的细胞活率均高于对照组的细胞活率;冻存后,本发明间充质干细胞注射液的下降幅度小,降低率为0. 10%/月,而对照组的下降幅度大,降低率为0. 38%/月,前者显著低于后者。 [0142] As can be seen from Table 5, 1,6,12 months after cryopreservation recovery charge between the present invention was clearly higher cell viability of living cells of mesenchymal stem cell injections; after cryopreservation, the present invention mesenchymal stem cell injection of a small decline, reducing the rate of 0.10% / month, and a large decrease in the control group, the reduction rate of 0.38% / month, the former is significantly lower than the latter.

[0143] 本实施例进一步验证了本发明间充质干细胞可以长时间维持细胞活率。 [0143] Example embodiment of the present invention further verified between mesenchymal stem cells may be maintained for a long time cell viability.

[0144] 综上,本发明冻存液冻可以在冻存时长时间维持细胞活率,冻存复苏后,细胞活率下降慢;既不含胎牛血清等不能直接临床输注的成分,也不含人血浆等存在安全隐患且成本极高的成分,因此,本发明的间充质干细胞注射液冻存效果极好,安全,成本低廉,有效期长,使用非常方便,具有良好的工业应用前景。 The [0144] Fully, frozen cryopreservation solution of the present invention can be maintained for a long time when cryopreserved cell viability after cryopreservation, cell viability decreased slowly; can not be directly infused ingredients contained neither clinical fetal calf serum, also excluding the presence of human plasma and other security risks, and the high cost of components, therefore, between the present invention mesenchymal stem cell cryopreservation excellent injection effect, safety and low cost, long shelf life, easy to use, has good prospects for industrial applications .

Claims (4)

1. 一种间充质干细胞冻存液,其特征在于:它包含如下体积百分比的成分: A mesenchymal stem cell freezing medium, characterized in that: it contains the following percentages by volume of the composition:
Figure CN103583511BC00021
2. 根据权利要求1所述的冻存液,其特征在于:它由如下体积百分比的成分组成: 2. The cryopreservation solution according to claim 1, characterized in that: it is the percentage by the following chemical composition by volume:
Figure CN103583511BC00022
3. -种间充质干细胞注射液,其特征在于:它包含权利要求1或2所述的间充质干细胞冻存液和间充质干细胞,间充质干细胞的浓度为1X10 5~1X108个/ml。 3. - species mesenchymal stem cell injection, characterized in that: it contains as claimed in claim mesenchymal stem cell cryopreservation and mesenchymal stem cells of claim 1 or 2, the mesenchymal stem cells at a concentration of 1X10 5 ~ 1X108 months / ml.
4. 一种制备权利要求3所述间充质干细胞注射液的方法,其特征在于:它包括如下步骤: (1) 按照权利要求1或2所述比例取勃脉力A液、18AA 5%复方氨基酸注射液、20%人血白蛋白和DMSO ; (2) 将步骤⑴勃脉力A液、18AA 5%复方氨基酸注射液和20%人血白蛋白混勾,加入间充质干细胞至细胞浓度为1 X 105~1 X 10 8个/ml,得细胞悬液; (3) 将步骤(l)DMSO加入步骤⑵所得的细胞悬液,即可。 The three mesenchymal stem cell injections 4. A method of preparing claims, characterized in that: it comprises the following steps: (1) according to claim 1 or 2 ratio was taken Plasmalyte A, 18AA 5% compound amino acid injection, 20% human serum albumin and DMSO; (2) the step ⑴ Plasmalyte A solution, 18AA 5% compound amino acid injection and mixed with 20% human serum albumin hook, between mesenchymal stem cells was added to the cells at a concentration of 1 X 105 ~ 1 X 10 8 / ml, and cell suspension obtained; (3) the step (l) DMSO added to the cell suspension obtained in step ⑵, can.
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