WO2012068710A1 - Methods for extracting mesenchymal stem cell from slight amount human adipose tissue and mass cultivation thereof - Google Patents
Methods for extracting mesenchymal stem cell from slight amount human adipose tissue and mass cultivation thereof Download PDFInfo
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- WO2012068710A1 WO2012068710A1 PCT/CN2010/002101 CN2010002101W WO2012068710A1 WO 2012068710 A1 WO2012068710 A1 WO 2012068710A1 CN 2010002101 W CN2010002101 W CN 2010002101W WO 2012068710 A1 WO2012068710 A1 WO 2012068710A1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- the invention belongs to the field of biomedicine, and particularly relates to a method for extracting mesenchymal stem cells from a trace amount of human adipose tissue and large-scale culture.
- Mesenchymal stem cells are a group of mesenchymal-derived pluripotent stem cells with self-renewal and multi-directional differentiation potential.
- Mesenchymal stem cells not only have the commonality of stem cells, namely the ability to self-renew, multi-directional differentiation and homing, but also have the ability to differentiate into many types of cells, which can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve.
- tissue cells such as liver, heart muscle and endothelium still have multi-directional differentiation potential after continuous subculture and cryopreservation. Animal experiments and preliminary clinical studies have shown that mesenchymal stem cells have low immunogenicity and immunosuppressive effects. Therefore, mesenchymal stem cells can be used as ideal seed cells for the repair of tissue and organ damage caused by aging and lesions.
- Mesenchymal stem cells are initially found in the bone marrow and are receiving increasing attention due to their multi-directional differentiation potential, support for hematopoiesis, and promotion of stem cell implantation, immune regulation, and self-replication.
- the content is very low, accounting for only 1/10 5 to 10 6 of the monocytes in the bone marrow, and collecting bone marrow brings great pain to the patients.
- a source tissue that is simple and rich in mesenchymal stem cells.
- Current research shows that adipose tissue contains a precursor cell population that can differentiate into adipocytes, bone cells, and chondrocytes.
- adipose stromal cells adipose-derived mesenchymal stem cells.
- adipose tissue can be obtained by liposuction of the abdomen. It is less injurious to the patient and relatively easy to obtain.
- adipose-derived mesenchymal stem cells are less susceptible to contamination by tumor cells and are easier to purify in vitro.
- Autologous cells, and transplanted cells are more easily accepted by the patient's own tissues, faster integration into their own tissue systems to play a role, avoiding a series of ethical issues and transplant side effects such as immune rejection of allogeneic cells.
- the general steps of extracting adipose-derived mesenchymal stem cells from fat include anesthesia and liposuction. Taking a large amount of human adipose tissue, digested with collagenase, subjected to gradient centrifugation, filtration, and then expanded culture in an in vitro culture system, wherein the in vitro culture system contains fetal calf serum; and a large area is required in the liposuction process. Local anesthesia, and a mouth in the liposuction, caused some trauma and pain to the patient.
- the present invention provides a method for extracting mesenchymal stem cells from microcalori fat tissue and large-scale cultivation to reduce patient suffering and satisfy clinical safety applications.
- a method for extracting mesenchymal stem cells from a trace of human adipose tissue comprising the following steps:
- step (1) the extracted adipose tissue is 5 ml.
- step (2) the phosphate is soaked in phosphate buffer solution, and the fat is washed by centrifugation at 1500 r/min, and digested with 0.1% type I collagenase in a water bath at 37 ° C for about 30 minutes, and the collagenase is diluted with a phosphate buffer and centrifuged to suspend the lower layer.
- the precipitate was filtered through a 100 mesh filter and centrifuged at 1500 r/m for 10 min to remove suspended fat cells and lipid droplets, and the supernatant was discarded, followed by in vitro culture.
- the method for large-scale cultivation of mesenchymal stem cells of the present invention mainly comprises the following steps:
- the culture system is STEMPRO MSC SFM serum-free medium containing 2 mM/ml glutamine and 100 U/ml cyan-streptomycin;
- the invention effectively reduces the suffering of the patient, eliminates the obstacle that the very thin patient cannot obtain the fat tissue, and avoids the use of the animal source product in the whole cell culture system, especially the serum product, which satisfies the needs of future clinical safety application.
- Figure 1 is a view showing the morphology of adipose-derived mesenchymal stem cells under an inverted coma microscope;
- Figure 2 is a schematic diagram showing the expression of surface markers of human adipose-derived mesenchymal stem cells by flow cytometry;
- Figure 3 is a graph showing the detection of adipogenic and osteogenic differentiation of cultured third-generation cells.
- STEMPRO MSC SFM serum-free medium cyanine-streptomycin, gentamicin, glutamine, platelet-derived growth factor, basic fibroblast growth factor, type I collagenase, trypsin, phosphate buffer, oil Red 0, alizarin red.
- the adipose tissue was washed with phosphate buffer solution and centrifuged at 1500 r/min, and digested with 0.1% type I collagenase in a water bath at 37 ° C for about 30 min.
- the collagenase was diluted with a phosphate buffer and centrifuged, and the lower layer was suspended and filtered through a 100 mesh screen. Centrifuge at 1500 r/m for 10 min to remove suspended fat cells and lipid droplets, discard the supernatant, add serum-free medium, pipette evenly, centrifuge at 1000 r/m for 5 min , discard the supernatant, and add complete medium A.
- the cells were evenly beaten, trypan blue staining, and after counting with a counting plate, the cells were seeded in a culture flask at a density of lx 10 5 /mL, and cultured at 37 ° C, 5% CO 2 , and saturated humidity.
- the unattached cells were discarded 24 to 48 hours and the same medium was added. After every 2 ⁇ 3d, the liquid was changed to P0 generation; when the cells reached 8 (90% confluence, trypsinized, passaged at a ratio of 1:3, and labeled as Pl.
- Morphological characteristics of adipose-derived mesenchymal stem cells The cultured cells were observed to have a fusiform fibroblast-like appearance under an inverted phase contrast microscope, which was partially swirled (as shown in Fig. 1).
- Phenotypic detection of human adipose-derived mesenchymal stem cells The expression of the surface markers of the spindle cells obtained above was detected by direct immunofluorescence, and the cells were phycoerythrin (PE) CD44 and fluorescein isothiocyanate (FITC). Flow detection was performed after CD34 labeling, and the flow cytometer was BD FACScan (Becton Dickinson:). The results indicate that this spindle cell expresses the surface marker CD44 of mesenchymal stem cells and does not express the surface marker CD34 of hematopoietic stem cells (as shown in Figure 2).
- Human adipose-derived mesenchymal stem cells differentiated into adipocytes and stained with oil red O.
- the adipogenic induction medium comprises: basal medium DMEM, 10% fetal bovine serum, 0.5 mM IBMX (isobulyl-l-methylxanthione), O. lmM indomethacin, luM dexamethasone. Oil red O staining was performed on days 6, 12, and 16, respectively.
- vacuoles with increased refractive power were filled throughout the cells for 12 days; oil red 0 staining found that about 60 to 80% of the cells in the culture were rich in fat droplets (as shown in Fig. 3A). This indicates that stem cell subsets can be induced to differentiate into adipocytes in vitro.
- Human adipose-derived mesenchymal stem cells differentiate into osteoblasts and stained with alizarin red for the third generation of human mesenchymal stem cells, cultured in 6-well plates, and replaced with osteogenic induction medium when the cells grow to about 80%.
- the components of the inducing solution include: basal medium DMEM, 10% fetal bovine serum, ⁇ -phosphoglycerol, O. lmM ascorbyl phosphate Vc, O. luM dexamethasone. The liquid was changed every three days, cultured for 3 weeks, and alizarin red staining was observed at 2 weeks and 3 weeks, respectively.
- serum-free culture system B STEMPRO MSC SFM serum-free medium containing 2 mM/ml of glutamine Amide, 10 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor,
Abstract
Methods for extracting mesenchymal stem cell from slight amount human adipose tissue and mass cultivation thereof are provided, wherein the method for extracting mesenchymal stem cell from slight amount human adipose tissue comprises: injecting lidocaine for local anesthesia in small area, carrying out aseptic extraction of the slight amount adipose tissue of a patient using a miniature suction needle; then soaking and centrifugally washing the adipose tissue by aseptic extraction using the phosphate buffer solution, digesting using collagenase, performing gradient centrifugation, filtering, and culturing in an in-vitro culturing system. The method effectively reduces the pain of the patient, resolves the problem that adipose tissue cannot be extracted from a very thin patient, avoids using animal source products in whole cell cultivation system, especially not using serum products, and meets the requirement of clinical safe application in the future.
Description
说 明 书 从微量人脂肪组织提取间充质干细胞及规模化培养的方法 Method for extracting mesenchymal stem cells from micro-human adipose tissue and large-scale culture method
(一） 技术领域 _ (1) Technical field _
本发明属于生物医学领域， 特别涉及一种从微量人脂肪组织提取 间充质干细胞及规模化培养的方法。 The invention belongs to the field of biomedicine, and particularly relates to a method for extracting mesenchymal stem cells from a trace amount of human adipose tissue and large-scale culture.
(二） 背景技术 (2) Background technology
间充质干细胞是一群中胚层来源的具有自我更新和多向分化潜 能的多能干细胞。间充质干细胞不仅具有干细胞的共性， 即自我更新、 多向分化和归巢的能力， 还具有向多种类型细胞分化的能力， 可以分 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， 动 物实验和初步的临床研究已显示间充质干细胞具有低免疫原性和免 疫抑制的作用， 所以间充质干细胞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。 Mesenchymal stem cells are a group of mesenchymal-derived pluripotent stem cells with self-renewal and multi-directional differentiation potential. Mesenchymal stem cells not only have the commonality of stem cells, namely the ability to self-renew, multi-directional differentiation and homing, but also have the ability to differentiate into many types of cells, which can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve. Various tissue cells such as liver, heart muscle and endothelium still have multi-directional differentiation potential after continuous subculture and cryopreservation. Animal experiments and preliminary clinical studies have shown that mesenchymal stem cells have low immunogenicity and immunosuppressive effects. Therefore, mesenchymal stem cells can be used as ideal seed cells for the repair of tissue and organ damage caused by aging and lesions.
间充质干细胞最初在骨髓中发现， 因其具有多向分化潜能、 支持 造血和促进干细胞植入、 免疫调控和自我复制等特点而日益受到人们 的关注。 然而就目前已比较成熟的骨髓间充质干细胞而言， 其含量非 常低， 仅占骨髓中单核细胞的 1/105〜106， 并且采集骨髓给患者带来极 大的痛苦， 取材不方便， 所以这就迫切需要寻找一种取材简单且间充 质干细胞丰富的源组织。 目前的研究表明脂肪组织中含有能分化为脂 肪细胞、 骨细胞、 软骨细胞的前体细胞群， 具有较强的增殖能力和多 分化潜能， 可称之为脂肪基质细胞或脂肪间充质干细胞。与骨髓相比， 脂肪组织可通过腹部的吸脂术获得，对患者损伤较小，获取相对容易， 更重要的是脂肪间充质干细胞不易受到肿瘤细胞污染， 更容易进行体 外净化， 病人可以用自体细胞， 并且移植细胞更易被患者自身组织所 接受， 更快的整合到自身组织系统以发挥作用， 避免了一系列的伦理 问题和异体细胞的免疫排斥等移植副反应。 Mesenchymal stem cells are initially found in the bone marrow and are receiving increasing attention due to their multi-directional differentiation potential, support for hematopoiesis, and promotion of stem cell implantation, immune regulation, and self-replication. However, in the case of the relatively mature bone marrow mesenchymal stem cells, the content is very low, accounting for only 1/10 5 to 10 6 of the monocytes in the bone marrow, and collecting bone marrow brings great pain to the patients. Convenient, so it is urgent to find a source tissue that is simple and rich in mesenchymal stem cells. Current research shows that adipose tissue contains a precursor cell population that can differentiate into adipocytes, bone cells, and chondrocytes. It has strong proliferative capacity and multi-differentiation potential, and can be called adipose stromal cells or adipose-derived mesenchymal stem cells. Compared with bone marrow, adipose tissue can be obtained by liposuction of the abdomen. It is less injurious to the patient and relatively easy to obtain. More importantly, adipose-derived mesenchymal stem cells are less susceptible to contamination by tumor cells and are easier to purify in vitro. Autologous cells, and transplanted cells are more easily accepted by the patient's own tissues, faster integration into their own tissue systems to play a role, avoiding a series of ethical issues and transplant side effects such as immune rejection of allogeneic cells.
目前从脂肪中提取脂肪间充质干细胞一般步骤包括麻醉抽脂获
取大量的人体脂肪组织， 用胶原酶消化， 经梯度离心、 过滤， 随后在 体外培养体系中进行扩增培养， 其中所述体外培养体系中含有胎牛 清； 并且抽脂过程中需要大面积的局部麻醉， 并在抽脂部位切一个口， 对病人造成了一定的创伤和痛苦， 为了获得大量的脂肪组织， 需要大 面积的抽脂手术， 导致患者抽脂面积较大， 手术时间较长， 出血较多 或导致发热现象， 如果后期护理不好容易引发炎症。 At present, the general steps of extracting adipose-derived mesenchymal stem cells from fat include anesthesia and liposuction. Taking a large amount of human adipose tissue, digested with collagenase, subjected to gradient centrifugation, filtration, and then expanded culture in an in vitro culture system, wherein the in vitro culture system contains fetal calf serum; and a large area is required in the liposuction process. Local anesthesia, and a mouth in the liposuction, caused some trauma and pain to the patient. In order to obtain a large amount of adipose tissue, a large area of liposuction is required, resulting in a large liposuction area and a long operation time. More bleeding or fever, if the late care is not easy to cause inflammation.
(三） 发明内容 (3) Invention content
本发明为了弥补现有技术的不足， 提供了一种减少病人痛苦、 满 足临床安全应用的从微量热脂肪组织提取间充质干细胞及规模化培 养的方法。 In order to make up for the deficiencies of the prior art, the present invention provides a method for extracting mesenchymal stem cells from microcalori fat tissue and large-scale cultivation to reduce patient suffering and satisfy clinical safety applications.
本发明是通过如下技术方案实现的： The invention is achieved by the following technical solutions:
一种从微量人脂肪组织提取间充质干细胞的方法， 主要包括如下 步骤： A method for extracting mesenchymal stem cells from a trace of human adipose tissue, comprising the following steps:
( 1 ) 微创抽脂， 注射利多卡因小面积的局部麻醉， 利用微型吸 引针无菌抽取患者微量脂肪组织； (1) minimally invasive liposuction, local anesthesia in a small area of lidocaine, and micro-absorbent tissue is used to aseptically extract trace adipose tissue from patients;
( 2 ) 脂肪间充质干细胞的提取， 采用磷酸盐缓冲液浸泡、 离心 洗涤无菌获取的脂肪组织， 用胶原酶消化， 经梯度离心、 过滤， 随后 在体外培养体系中进行培养。 (2) Extraction of adipose-derived mesenchymal stem cells, soaking the adipose tissue obtained by immersion in a phosphate buffer solution, centrifuging, and digesting with collagenase, centrifuging, filtering, and then culturing in an in vitro culture system.
步骤 （1 ) 中， 抽取的脂肪组织为 5ml。 In step (1), the extracted adipose tissue is 5 ml.
步骤 （2 ) 中， 采用磷酸盐缓冲液浸泡， 1500r/min离心洗涤脂肪 至， 用 0. 1% I型胶原酶 37 °C水浴消化约 30min， 加磷酸缓冲液稀释胶 原酶并离心， 悬浮下层沉淀， 100 目滤网过滤， 1500r/m离心 10min， 以去除悬浮的脂肪细胞及脂滴， 弃上清液， 随后进行体外培养。 In step (2), the phosphate is soaked in phosphate buffer solution, and the fat is washed by centrifugation at 1500 r/min, and digested with 0.1% type I collagenase in a water bath at 37 ° C for about 30 minutes, and the collagenase is diluted with a phosphate buffer and centrifuged to suspend the lower layer. The precipitate was filtered through a 100 mesh filter and centrifuged at 1500 r/m for 10 min to remove suspended fat cells and lipid droplets, and the supernatant was discarded, followed by in vitro culture.
本发明间充质干细胞规模化培养的方法， 主要包括如下步骤： The method for large-scale cultivation of mesenchymal stem cells of the present invention mainly comprises the following steps:
( 1 )原代培养，培养体系为 STEMPRO MSC SFM 无血清培养基， 含有 2mM/ml的谷氨酰胺和 100U/ml的青-链霉素； (1) Primary culture, the culture system is STEMPRO MSC SFM serum-free medium containing 2 mM/ml glutamine and 100 U/ml cyan-streptomycin;
( 2 ) 细胞鉴定， 流式检测细胞表面抗原 CD44和 CD34的表达， 体外分化实验检测脂肪间充质干细胞多向分化潜能， 包括像脂肪细胞 的分化和成骨细胞的分化； (2) Cell identification, flow detection of cell surface antigens CD44 and CD34 expression, in vitro differentiation assay to detect multipotential differentiation potential of adipose-derived mesenchymal stem cells, including differentiation of adipocytes and differentiation of osteoblasts;
( 3 ) 规模化扩增， 采用 STEMPRO MSC SFM无血清培养体系，
含有 2mM/ml的谷氨酰胺、 10ng/ml的血小板衍生生长因子、 10ng/ml 的碱性成纤维细胞生长因子和 0.5 μ g/ml的庆大霉素。 (3) Scale-up amplification, using STEMPRO MSC SFM serum-free culture system, Contains 2 mM/ml glutamine, 10 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor, and 0.5 μg/ml gentamicin.
本发明有效减少了病人的痛苦， 排除了非常瘦的患者无法获取脂 肪组织的障碍， 整个细胞培养体系中尽量避免了使用动物源制品， 特 别是没有血清制品， 满足了将来临床安全应用的需求。 The invention effectively reduces the suffering of the patient, eliminates the obstacle that the very thin patient cannot obtain the fat tissue, and avoids the use of the animal source product in the whole cell culture system, especially the serum product, which satisfies the needs of future clinical safety application.
(四） 附图说明 (4) Description of the drawings
下面结合附图对本发明作进一步的说明。 The invention will now be further described with reference to the accompanying drawings.
图 1为倒置枏差显微镜下脂肪间充质干细胞的形态图； Figure 1 is a view showing the morphology of adipose-derived mesenchymal stem cells under an inverted coma microscope;
图 2为流式检测人脂肪间充质干细胞表面标志的表达示意图； 图 3为培养的第三代细胞成脂和成骨诱导分化染色检测图。 Figure 2 is a schematic diagram showing the expression of surface markers of human adipose-derived mesenchymal stem cells by flow cytometry; Figure 3 is a graph showing the detection of adipogenic and osteogenic differentiation of cultured third-generation cells.
(五） 具体实施方式 (5) Specific implementation methods
实施例： Example:
' 1.试剂 ' 1. Reagent
STEMPRO MSC SFM无血清培养基、 青-链霉素， 庆大霉素、 谷 氨酰胺、 血小板衍生生长因子、 碱性成纤维细胞生长因子、 I 型胶原 酶、 胰酶、 磷酸盐缓冲液、 油红 0、 茜素红。 STEMPRO MSC SFM serum-free medium, cyanine-streptomycin, gentamicin, glutamine, platelet-derived growth factor, basic fibroblast growth factor, type I collagenase, trypsin, phosphate buffer, oil Red 0, alizarin red.
2.脂肪间充质干细胞的分离提取 2. Separation and extraction of adipose-derived mesenchymal stem cells
采用磷酸盐缓冲液浸泡、 1500r/min离心洗涤脂肪组织，用 0.1% I 型胶原酶 37°C水浴消化约 30min， 加磷酸缓液稀释胶原酶并离心， 悬 浮下层沉淀， 100 目筛网过滤， 1500 r/m离心 10 min， 以去除悬浮的 脂肪细胞及脂滴， 弃上清液， 加入无血清培养液， 吹打均匀， 1000 r/m 离心 5min; 弃上清液， 加入完全培养基 A， 吹打均匀， 台盼蓝染色， 用计数板计数后，按 l x l05/mL的密度将细胞接种于培养瓶中，在 37°C、 5%C02、 饱和湿度条件下培养。 The adipose tissue was washed with phosphate buffer solution and centrifuged at 1500 r/min, and digested with 0.1% type I collagenase in a water bath at 37 ° C for about 30 min. The collagenase was diluted with a phosphate buffer and centrifuged, and the lower layer was suspended and filtered through a 100 mesh screen. Centrifuge at 1500 r/m for 10 min to remove suspended fat cells and lipid droplets, discard the supernatant, add serum-free medium, pipette evenly, centrifuge at 1000 r/m for 5 min , discard the supernatant, and add complete medium A. The cells were evenly beaten, trypan blue staining, and after counting with a counting plate, the cells were seeded in a culture flask at a density of lx 10 5 /mL, and cultured at 37 ° C, 5% CO 2 , and saturated humidity.
24~48小时弃去未贴壁的细胞，并补充相同的培养液。以后每 2~3d 半换液， 记为 P0代； 待细胞达到 8( 90%汇合时， 胰蛋白酶消化， 按 1 :3比例传代， 并标记为 Pl。 The unattached cells were discarded 24 to 48 hours and the same medium was added. After every 2~3d, the liquid was changed to P0 generation; when the cells reached 8 (90% confluence, trypsinized, passaged at a ratio of 1:3, and labeled as Pl.
3、 脂肪间充质干细胞的鉴定 3. Identification of adipose-derived mesenchymal stem cells
( 1 ) 脂肪间充质干细胞的形态特征： 倒置相差显微镜下观察培 养的细胞呈梭形的成纤维细胞样， 局部呈漩涡状 （如图 1所示）。
( 2 ) 人脂肪间充质干细胞的表型检测： 用直接免疫荧光法检测 以上得到的梭形细胞的表面标志的表达， 细胞用藻红蛋白 （ PE ) CD44和异硫氰酸荧光素（ FITC ) CD34标记后进行流式检测， 流式 细胞仪为 BD FACScan ( Becton Dickinson ：)。 结果表明这种梭形细胞 表达间充质干细胞的表面标志 CD44,不表达造血干细胞的表面标志 CD34 (如图 2所示）。 (1) Morphological characteristics of adipose-derived mesenchymal stem cells: The cultured cells were observed to have a fusiform fibroblast-like appearance under an inverted phase contrast microscope, which was partially swirled (as shown in Fig. 1). (2) Phenotypic detection of human adipose-derived mesenchymal stem cells: The expression of the surface markers of the spindle cells obtained above was detected by direct immunofluorescence, and the cells were phycoerythrin (PE) CD44 and fluorescein isothiocyanate (FITC). Flow detection was performed after CD34 labeling, and the flow cytometer was BD FACScan (Becton Dickinson:). The results indicate that this spindle cell expresses the surface marker CD44 of mesenchymal stem cells and does not express the surface marker CD34 of hematopoietic stem cells (as shown in Figure 2).
( 3 ) 人脂肪间充质干细胞向脂肪细胞分化及油红 O染色检测 细胞传至第 3代时将其以 2 X 107/L的细胞数转入成脂诱导培养 基中进行培养。 诱导培养后不同时间段， 对成脂诱导组进行油红 O脂 肪颗粒染色。 成脂诱导培养基包含： 基础培养基 DMEM, 10%胎牛血 清、 0.5mM IBMX ( isobulyl-l-methylxanthione )、 O. lmM消炎痛、 luM 地塞米松。 在第 6天、 第 12 天和第 16 天分别进行油红 O 染色。 结果显示： 培养 12 天可见到折光度增加的液泡充满整个细胞； 油红 0 染色发现培养中约 60〜80 %细胞富含脂肪滴 （如图 3A所示）。 说 明干细胞亚群可在体外诱导分化成脂肪细胞。 (3) Human adipose-derived mesenchymal stem cells differentiated into adipocytes and stained with oil red O. When the cells were passed to the third passage, they were transferred to adipogenic medium in 2 x 10 7 /L cells for culture. Oil red O fat particles were stained in the adipogenic induction group at different time points after induced culture. The adipogenic induction medium comprises: basal medium DMEM, 10% fetal bovine serum, 0.5 mM IBMX (isobulyl-l-methylxanthione), O. lmM indomethacin, luM dexamethasone. Oil red O staining was performed on days 6, 12, and 16, respectively. The results showed that: vacuoles with increased refractive power were filled throughout the cells for 12 days; oil red 0 staining found that about 60 to 80% of the cells in the culture were rich in fat droplets (as shown in Fig. 3A). This indicates that stem cell subsets can be induced to differentiate into adipocytes in vitro.
( 4 ) 人脂肪间充质干细胞向骨细胞分化及茜素红染色检测 取第三代人间充质干细胞， 培养在 6孔板中， 待细胞长到 80%左 右时换用成骨诱导液培养， 诱导液的成分包含： 基础培养基 DMEM, 10%胎牛血清、 ΙΟΜιηβ-磷酸甘油、 O. lmM抗坏血酸磷酸盐 Vc、 O. luM 地塞米松。 每三天换液， 培养 3周， 于 2周、 3周分别进行茜素红染 色观察。 结果显示： 成骨诱导培养中细胞形态由原来的梭形变成了立 方形， 随着细胞生长密度的增长形成多层的结节结构， 而对照组中细 胞仍是梭形且呈单层生长； 2 周之后已有矿化结节形成了， 3 周后结 节比较多， 染色比较明显 （如图 3B所示）。 说明干细胞亚群可在体外 诱导分化为成骨细胞。 (4) Human adipose-derived mesenchymal stem cells differentiate into osteoblasts and stained with alizarin red for the third generation of human mesenchymal stem cells, cultured in 6-well plates, and replaced with osteogenic induction medium when the cells grow to about 80%. The components of the inducing solution include: basal medium DMEM, 10% fetal bovine serum, ΙΟΜιηβ-phosphoglycerol, O. lmM ascorbyl phosphate Vc, O. luM dexamethasone. The liquid was changed every three days, cultured for 3 weeks, and alizarin red staining was observed at 2 weeks and 3 weeks, respectively. The results showed that the morphology of the cells in the osteogenic induction culture changed from the original fusiform to cuboid, and the multi-layered nodular structure was formed as the cell growth density increased, while the cells in the control group were still fusiform and monolayer-grown. After 2 weeks, mineralized nodules were formed, and after 3 weeks, there were more nodules, and the staining was more obvious (as shown in Figure 3B). This indicates that stem cell subsets can be induced to differentiate into osteoblasts in vitro.
4.人脂肪间充质干细胞的规模化扩增 4. Large-scale expansion of human adipose-derived mesenchymal stem cells
培养的原代 （P0代） 细胞达到 80〜90%汇合时， 胰蛋白酶消化， 按 1 :3比例传代， 采用无血清培养体系 B(STEMPRO MSC SFM 无血 清培养基， 含有 2mM/ml 的谷氨酰胺、 10ng/ml 的血小板衍生生长因 子、 10ng/ml的碱性成纤维细胞生长因子、 0.5 g/ml庆大霉素)进行扩
增， 其扩增速度比用培养体系 A快， 细胞达到 90%汇合， 按 1 :3传代， 一般细胞 2〜3天传代一次； 而培养体系 A中的细胞按同样比例传代， 细胞一般 3~4天传代一次。 3周就可得到 109个细胞， 细胞状态良好。
Cultured primary (P0 generation) cells reached 80 to 90% confluence, trypsinized, passaged at a ratio of 1:3, using serum-free culture system B (STEMPRO MSC SFM serum-free medium containing 2 mM/ml of glutamine Amide, 10 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor, 0.5 g/ml gentamicin) Increase, the amplification rate is faster than that of culture system A, the cells reach 90% confluence, pass through 1:3, and the cells are passaged once every 2~3 days; while the cells in culture system A are passaged in the same proportion, the cells are generally 3~ Passed once every 4 days. 10 9 cells were obtained in 3 weeks, and the cells were in good condition.
Claims
1. 一种从微量人脂肪组织提取间充质干细胞的方法， 其特征为， 主 要包括如下步骤： （1 ) 微创抽脂， 注射利多卡因小面积的局部麻 醉， 利用微型吸引针无菌抽取患者微量脂肪组织； （2 ) 脂肪间充 质干细胞的提取， 采用磷酸盐缓冲液浸泡、 离心洗涤无菌获取的 脂肪组织， 用胶原酶消化， 经梯度离心、 过滤， 随后在体外培养 体系中进行培养。 A method for extracting mesenchymal stem cells from a trace amount of human adipose tissue, which comprises the following steps: (1) minimally invasive liposuction, local anesthesia injecting a small area of lidocaine, using a micro-sucking needle aseptically Extracting adipose tissue from patients; (2) extracting adipose-derived mesenchymal stem cells, soaking and aspirating the adipose tissue obtained by immersion in a phosphate buffer solution, digesting with collagenase, centrifuging, filtering, and then in vitro culture system Cultivate.
2. 根据权利要求 1 所述的从微量人脂肪组织提取间充质干细胞的方 法， 其特征在于： 步骤 （1 ) 中， 抽取的脂肪组织为 5ml。 The method for extracting mesenchymal stem cells from a trace amount of human adipose tissue according to claim 1, wherein in step (1), the extracted adipose tissue is 5 ml.
3. 根据权利要求 1 或 2所述的从微量人脂肪组织提取间充质干细胞 的方法， 其特征在于： 步骤 （2 ) 中， 采用磷酸盐缓冲液浸泡， 1500r/min离心洗涤脂肪至， 用 0. 1% I型胶原酶 37 °C水浴消化约 30min , 加磷酸缓冲液稀释胶原酶并离心， 悬浮下层沉淀， 100 目 滤网过滤， 1500r/m离心 10min， 以去除悬浮的脂肪细胞及脂滴， 弃上清液， 随后进行体外培养。 The method for extracting mesenchymal stem cells from a trace amount of human adipose tissue according to claim 1 or 2, wherein: in step (2), the phosphate is immersed in a phosphate buffer solution, and the fat is washed by centrifugation at 1500 r/min. 0. 1% type I collagenase was digested in a 37 °C water bath for about 30 minutes. The collagenase was diluted with phosphate buffer and centrifuged. The lower layer sediment was suspended, filtered through a 100 mesh filter, and centrifuged at 1500 r/m for 10 min to remove suspended fat cells and lipids. Drop, discard the supernatant, and then culture in vitro.
4. 根据权利要求 1 所述的间充质干细胞规模化培养的方法， 其特征 为，主要包括如下步骤：（1 )原代培养，培养体系为 STEMPRO MSC SFM 无血清培养基， 含有 2mM/ml 的谷氨酰胺和 100U/ml 的青- 链霉素； （2 ) 细胞鉴定， 流式检测细胞表面抗原 CD44和 CD34的 表达， 体外分化实验检测脂肪间充质干细胞多向分化潜能， 包括 像脂肪细胞的分化和成骨细胞的分化； （3 ) 规模化扩增， 采用 STEMPRO MSC SFM无血清培养体系，含有 2mM/ml的谷氨酰胺、 10ng/ml 的血小板衍生生长因子、 10ng/ml 的碱性成纤维细胞生长 因子和 0.5 μ g/ml的庆大霉素。 The method for large-scale culture of mesenchymal stem cells according to claim 1, characterized in that it mainly comprises the following steps: (1) primary culture, the culture system is STEMPRO MSC SFM serum-free medium, containing 2 mM/ml Glutamine and 100 U/ml cyan-streptomycin; (2) cell identification, flow cytometry detection of cell surface antigens CD44 and CD34 expression, in vitro differentiation assay for detection of multi-directional differentiation potential of adipose-derived mesenchymal stem cells, including fat Differentiation of cells and differentiation of osteoblasts; (3) Scale-up, using STEMPRO MSC SFM serum-free culture system containing 2 mM/ml glutamine, 10 ng/ml platelet-derived growth factor, 10 ng/ml alkali Fibroblast growth factor and gentamicin at 0.5 μg/ml.
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| CN102732477B (en) * | 2012-06-15 | 2013-06-19 | 江苏瑞思坦生物科技有限公司 | Human adipose-derived stem cell serum-free basic medium |
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| CN103251649B (en) * | 2013-04-17 | 2018-10-19 | 冯文峰 | The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder |
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