WO2017059566A1 - 一种快速分离脂肪间质细胞的组合物 - Google Patents

一种快速分离脂肪间质细胞的组合物 Download PDF

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WO2017059566A1
WO2017059566A1 PCT/CN2015/091449 CN2015091449W WO2017059566A1 WO 2017059566 A1 WO2017059566 A1 WO 2017059566A1 CN 2015091449 W CN2015091449 W CN 2015091449W WO 2017059566 A1 WO2017059566 A1 WO 2017059566A1
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cells
adipose
stromal cells
composition
trypsin
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PCT/CN2015/091449
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French (fr)
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王耀贤
陈崇桓
何美泠
张瑞根
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高雄医学大学
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Priority to US15/766,013 priority Critical patent/US20180298342A1/en
Priority to PCT/CN2015/091449 priority patent/WO2017059566A1/zh
Priority to EP15905664.7A priority patent/EP3360957A4/en
Priority to JP2018516145A priority patent/JP6781752B2/ja
Priority to CN201580083577.6A priority patent/CN108138140A/zh
Publication of WO2017059566A1 publication Critical patent/WO2017059566A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)

Definitions

  • the present invention relates to a composition for isolating adipose stromal cells and a method for rapidly separating adipose stromal cells
  • Adipose-derived stem cells are an adult stem cell widely used in tissue engineering and regenerative medicine, and have multi-directional differentiation potential like bone marrow mesenchymal stem cells.
  • ADSCs Adipose-derived stem cells
  • the general method is to remove the tissue and then smash it and use collagenase to decompose the extracellular matrix. Its single cells are released from the tissue. Tissue decomposition takes about 12 hours or more (usually standing overnight). In addition to time consumption, leaving cells in an enzyme environment for a long time is not conducive to cell survival, often resulting in unstable or even dead cells, reducing the number of cells obtained.
  • the cells After removing mature cells such as red blood cells by mechanical and enzymatic treatment, the cells were cultured using a medium containing 10% fetal bovine serum.
  • the disadvantage of this method of culturing stem cells is that the method is complicated, the number of stem cells extracted is small, the purity is not high, and the proliferation of the passage is slow.
  • the non-fat single cells obtained by the liposuction technique obtained by the liposuction technique are called the Stromal Vascular Fraction (SVF), and the single cells are fast, but the liposuction probe is used.
  • SVF Stromal Vascular Fraction
  • Ultrasonic shock mode dissolves fat, which is extremely harmful to cells and affects cell survival rate.
  • mesenchymal stem cells can be used as a source of cells for bone regeneration and repair. Therefore, transplantation of autologous stem cells, especially autologous transplantation of adult adipose stem cells, has become a trend in the development of stem cell transplantation in the future.
  • separation from autologous tissue Purification of mesenchymal stem cells requires quite complicated procedures and is time consuming. If the mesenchymal stem cells are enlarged to a sufficient number of cells to be used in vitro, it is required to perform in a Good Laboratory Practice (GLP) laboratory to prolong the time of in vitro operation and reduce the chance of cells being infected by bacteria or viruses.
  • GLP Good Laboratory Practice
  • the term "plurality” is used to describe the number of components or units of the invention. This term should be understood to mean two or more unless expressly stated otherwise.
  • the present invention provides a composition and method for reducing patient suffering, satisfying clinical safety applications, and rapidly separating interstitial cells in a short time.
  • the present invention provides a composition for isolating adipose stromal cells comprising a type I collagenase at a concentration of 0.5-8% (v/v); a trypsin having a concentration of 0.1 -0.6% (v/v); and a metal ion chelating agent at a concentration of 0.01-0.2% (v/v).
  • metal ion chelating agent as used in the present invention is selected from ethylene diamine tetraacetic acid. (Ethylenediaminetetraacetic acid, EDTA) or its sodium salt, ethylene glycol tetraacetic acid (EGTA) or its sodium salt, diethyl triamine pentaacetic acid (DTPA) or its sodium Salt, polyphosphate, organophosphate, phosphate, polyacrylate, organophosphate, sodium gluconate, or a mixture of these.
  • EDTA ethylene diamine tetraacetic acid
  • EGTA ethylene glycol tetraacetic acid
  • DTPA diethyl triamine pentaacetic acid
  • composition for isolating adipose stromal cells of the present invention wherein the metal ion chelating agent is EDTA.
  • the composition for isolating adipose stromal cells of the present invention comprises the first type collagenase 2-4% (v/v) and trypsin 0.1-0.3% (v /v) and EDTA 0.01-0.1% (v/v).
  • a composition for isolating adipose stromal cells of the invention is sterile.
  • the composition for isolating adipose stromal cells of the present invention has the effect of allowing the adipose stromal cells to be sufficiently freed while protecting the cells from damage.
  • the present invention further provides a method for isolating adipose stromal cells, comprising the steps of: (a) obtaining an adipose tissue; (b) adding the composition for isolating the adipose stromal cells of the present invention to homogenize and reacting, thereby obtaining a a digested tissue mixture; wherein the composition comprises a type I collagenase of 0.5-8% (v/v); a trypsin of 0.1-0.6% (v/v); and a metal ion chelating agent 0.01-0.2 %(v/v); (c) centrifuging the digested tissue mixture of step (b) to remove impurities to obtain a filtrate containing adipose stromal cells; (d) adding a low-profile solution to step (c) The filtrate is reacted to obtain adipose stromal cell filtrate from which blood cells are removed; and (e) the filtrate of step (d) is neutralized
  • the adipose tissue is from a body; wherein the individual comprises an animal or a human.
  • the metal ion chelating agent is EDTA.
  • the first type of collagenase has a concentration of 2-4% (v/v), a trypsin concentration of 0.1-0.3% (v/v), and an EDTA concentration of 0.01- 0.1% (v/v).
  • the method of isolating adipose stromal cells is sterile.
  • the method for isolating adipose stromal cells of the present invention further has the feature that the adipose tissue can be sufficiently digested within a total reaction time of one hour, and the adipose stromal cells are more easily obtained.
  • the invention provides a method of isolating adipose stromal cells which is capable of isolating at least one million adipose stromal cells in 5 grams of adipose tissue.
  • the method for isolating adipose stromal cells of the present invention wherein the adipose stromal cells are differentiated into adipocytes, hematopoietic cells, vascular endothelial cells, osteoblasts, chondroblasts, and neural cells. Or epithelial cells.
  • the method of the present invention for isolating adipose stromal cells wherein the adipose stromal cells are differentiated into adipocytes, osteoblasts or chondroblasts.
  • the method of isolating adipose stromal cells of the present invention has the effect of allowing the adipose stromal cells to be sufficiently freed while protecting the cells from damage.
  • Fig. 1 is a flow chart showing an embodiment of a method for isolating adipose stromal cells of the present invention.
  • Figure 2 shows the results of the number of mesenchymal cells isolated from the inner thigh of SD rats by subcutaneous extraction of 5 g of adipose tissue within one hour of different concentrations of enzyme.
  • Figure 3 shows that 5 grams of adipose tissue is taken subcutaneously inside the thigh of the guinea pig, and the ratio of enzymes at different concentrations The number of interstitial cells that were formulated to be separated within one hour.
  • Figure 4 shows the analysis of the differentiation ability of the extracted adipose stromal cells by the selected adipose stem cells.
  • Figure 5 shows the cell surface biomarker expression of isolated adipose stem cells
  • Figure 6 shows the effect of different enzyme formulation concentrations on the survival of mesenchymal cells.
  • Figure 7 shows a comparison of the contents of the inventive compositions in place of different enzyme species.
  • the enzyme formulation of the present invention is applied to the rapid interstitial cell separation process of adipose tissue of rat Sprague-Dawley rat (SD rats) and guinea pig (Genia pig). schematic diagram.
  • adipose tissue was taken from the inner thigh of SD rats and guinea pigs, and an equal amount (about 5 mL) of the enzyme formulation buffer solution of the present invention (0.5-3% (v/v) type I collagenase) was added.
  • the upper layer of fat, connective tissue or unsmashed tissue was removed, washed with PBS (5 mL), centrifuged (300 ⁇ g) for 10 minutes, and then added in equal amounts (5 mL).
  • the low-tension solution was allowed to stand at room temperature for 5 minutes to remove the blood cells, and then neutralized by adding 10 mL of PBS buffer solution. After 10 minutes at 3,000 rpm, the solution was removed, and the lowermost cells were resuspended in 1 mL of PBS, and the number of cells was counted. The total reaction time is within one hour.
  • Example 2 20 ⁇ L of the adipose stromal cells sample isolated in Example 1 was taken out, and 20 ⁇ L of Trypan blue was added thereto to uniformly mix and stain, and then dropped into a cell counting dish to calculate surviving cells (non-stained cells).
  • adipose tissue was taken subcutaneously inside the thigh of SD rats, and the number of mesenchymal cells separated in one hour by different concentrations of enzyme was formulated.
  • the results showed that the enzyme formula (code G) was 3% (v/v) type I collagenase, 0.1% (v/v) trypsin (Trypsin), and 0.02% (v/v) EDTA.
  • the number of single cells extracted in one hour was the highest, and about 9 ⁇ 10 6 cells/mL of interstitial cells could be isolated.
  • Figure 3 shows the number of mesenchymal cells isolated from the inner side of the thigh of the guinea pig by 5 g of adipose tissue and separated in one hour by different enzyme ratios. The results showed that 3-4% (v/v) type I collagenase, 0.1% (v/v) trypsin (Trypsin) and 0.02% (v/v) EDTA enzyme formula (codes G, H) were The number of single cells extracted in one hour was the highest, and about 8 ⁇ 10 6 to 8.5 ⁇ 10 6 cells/mL of mesenchymal cells could be isolated.
  • Example 3 The differentiation ability of selected adipose stem cells
  • mesenchymal stem cells contain three characteristics:
  • the cells must be attached to the cell culture dish;
  • the surface antigen needs to express CD105, CD73, or CD90, but does not exhibit CD45, CD34, CD14 or CD11b, CD79a or CD19, or HLA-DR;
  • Mesenchymal stem cells need to have the characteristics of differentiation into adipocytes, osteoblasts and chondrocytes after induction.
  • Adipose-derived mesenchymal stem cells can be screened after one week of culture. As shown in (a) of Fig. 4, the isolated adipose-derived mesenchymal stem cells are adherent cells.
  • the analysis of the isolated adipose stromal cells by flow cytometry also showed that the expression of CD271, CD73 and CD90 on the cell surface was significantly higher than that of the control group, and the expression of CD34 was lower than that of the control group, as shown in Fig. 5. . Therefore, it is also known that the isolated adipose stromal cells belong to mesenchymal stem cells. The ability of the mesenchymal stem cells to induce differentiation into adipocytes, osteoblasts and chondrocytes is further examined below.
  • the adipose-derived mesenchymal stem cells isolated are cultured for two weeks in different mediums for differentiation (Adipo-medium: medium for inducing differentiation into adipocytes; Osteo-medium: medium for inducing differentiation into osteoblasts) And Chondro-medium: a medium that induces differentiation into chondrocytes; among them, Adipo-medium: DMEM medium (Dulbecco's Modified Eagle Medium), 10% fetal bovine serum (Fetal Bovine Serum FBS), 1% penicillin/streptomycin (penicillin/streptomycin), 500 ⁇ M 3-isobutyl-1-methylxanthine IBMX, 1 ⁇ M dexamethasone, 1 ⁇ M indomethasin, 10 ⁇ g/mL Insulin; Chondro-medium: DMEM medium (Dulbecco's Modified Eagle Medium), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 50
  • glycosaminoglycan GAG
  • the ability of mesenchymal stem cells to differentiate into chondrocytes was evaluated by the measurement of glycosaminoglycan (GAG), which was detected by Alcian blue staining.
  • GAG glycosaminoglycan
  • the old medium was removed and fixed with 10% formalin or 4% paraformaldehyde for 10 minutes. Wash twice with distilled water (3 mL). It was then shaken with 3% Acetic acid (3 mL) for 5 minutes. After removing all the liquid, erzon blue (1%) was added to the pan, shaken for 15 minutes, the dye was removed, and washed 2-3 times with distilled water (3 mL), and photographed was taken.
  • Elson blue stains the extracellular matrix GAG, which is blue in color and aggregates in cells, which is characteristic of chondrocytes.
  • the ability of mesenchymal stem cells to differentiate into adipocytes was assessed by staining oil droplets in adipocytes with Oil Red O. After two weeks of Adipo-medium culture, the old medium was removed and fixed in 10% formalin or 4% polyoxymethylene for 10 minutes. Wash twice with distilled water (3 mL). After removing all the liquid, Oil Red O (0.5%) was added to the tray, and the mixture was shaken for 10 minutes. The dye was removed and washed 2-3 times with distilled water (3 mL), and distilled water was added thereto, and photographing was carried out.
  • the oil droplets in the stained cells are red dots and are characteristic of fat cells.
  • osteogenic stem cells isolated by induced adipose-derived stem cells was examined by Alizarin-red staining. After two weeks of incubation in Osteo-medium, the old medium was removed and fixed in 4% formalin for 15 minutes. Wash twice with distilled water (3 mL). After removing all the liquid, a 2% blush solution was added to the tray, and the reaction was carried out at room temperature for 20 minutes. After removing the dye, it was washed 2-3 times with distilled water (3 mL) and photographed.
  • the calcium deposit produced by the stained cells is red dot-like and is characteristic of osteoblasts.
  • Adipose mesenchymal stem cells can be screened after one week.
  • the cytotoxicity test was carried out with the following three enzyme formulations for the isolation of the preferred adipose stromal cells disclosed in Example 1:
  • the formulation of this example does not have the same enzyme formula as 3% (v/v) type I collagenase, 0.1% (v/v) trypsin and 0.02% (v/v) EDTA. Get a similar number of cells. Therefore, the enzyme formulation of 3% (v/v) type I collagenase, 0.1% (v/v) trypsin and 0.02% (v/v) EDTA is irreplaceable in the rapid isolation of adipose stromal cells.

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Abstract

一种分离脂肪间质细胞的组合物，其包含一第一型胶原蛋白酶为0.5-8％(v/v)；一胰蛋白酶为0.1-0.6％(v/v)；及一金属离子螯合剂0.01-0.2％(v/v)。一种分离脂肪间质细胞的方法，该方法包括取得一脂肪组织；以上述组合物处理该脂肪组织；离心该脂肪组织；及分离该脂肪组织以获得脂肪间质细胞。该组合物和方法有助于未来在手术室中短时间快速分离间质细胞并运用于再生医学。

Description

一种快速分离脂肪间质细胞的组合物 技术领域
本发明是关于一种分离脂肪间质细胞的组合物及快速分离脂肪间质细胞的方法
背景技术
脂肪干细胞(adipose-derived stem cells，ADSCs)是目前广泛应用于组织工程及再生医学领域的一种成体干细胞，与骨髓间充质干细胞一样具有多向分化潜能。目前实验室常用的ADSCs分离培养方法是从脂肪组织中分离获取细胞，该方式已沿用数十年，通用方式为组织取下后将其剪碎并佐以胶原蛋白酶(collagenase)分解细胞外基质使其单细胞从组织释出。组织分解约需十二小时以上(通常静置过夜)。除了时间消耗外，长时间让细胞处于酵素环境并不利于细胞存活，常造成细胞生理状态不稳定甚至死亡减少细胞获得数量。经机械和酶处理方法除去红细胞等成熟细胞后，利用含10％胎牛血清的培养基进行培养。这种培养干细胞方法的缺点是方法复杂，提取的干细胞数量少，纯度不高，继代增殖缓慢。
坊间使用抽脂技术取得的液态脂肪组织佐以胶原蛋白酶分解离心后取得的非脂肪单细胞称为间质血管层细胞(Stromal Vascular Fraction，SVF)，取得单细胞虽然快速，但抽脂用探头使用超音波震荡方式溶脂吸出，对细胞伤害极大，影响细胞存活率。
近年来在再生医学，特别是骨科学研究强调，间质干细胞可运用于骨再生与修复的细胞来源，因此自体干细胞的移植，特别是成体脂肪干细胞的自体移植成为未来干细胞移植研发的趋势。然而，从自体组织中分离与 纯化间质干细胞需要相当复杂的操作程序且耗时。如在自体外将间质干细胞放大到足够使用的细胞数量，则需要优良实验室操作规范(Good Laboratory Practice，GLP)实验场所进行，延长体外操作的时间并降低细胞受细菌或病毒感染的机率。前述是干细胞在临床上运用的限制因素，本发明将有助于未来在手术室中短时间快速分离间质细胞并运用于再生医学。
发明内容
除非本文另外界定，否则本发明所用的科学及技术术语应具有一般熟习此项技术者通常所理解的含义。该等术语的含义及范畴应为清晰的；然而，在任何潜在歧义的情况下，本文所提供的定义优于任何辞典或外在定义。
本文中，术语“复数个”是用以描述本发明的组件或单元的数量。此用语除非明确另有所指，否则应理解为两个以上。
本文中的用语“一”或“一种”是用以叙述本发明的组件及成分。此术语仅为了叙述方便及给予本发明的基本观念。此叙述应被理解为包括一种或至少一种，且除非明显地另有所指，表示单数时亦包括复数。
本文中的用语“或”其意同“及/或”。
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、满足临床安全应用及短时间快速分离间质细胞的组合物及其方法。
本发明提供一种分离脂肪间质细胞的组合物，其包含一第一型胶原蛋白酶(type I collagenase)，浓度为0.5-8％(v/v)；一胰蛋白酶(Trypsin)，浓度为0.1-0.6％(v/v)；及一金属离子螯合剂，浓度为0.01-0.2％(v/v)。
本发明中所使用的术语“金属离子螯合剂”是选自乙烯二胺四乙酸 (Ethylenediaminetetraacetic acid，EDTA)或其钠盐、乙二醇双氨乙基醚四乙酸(ethylene glycol tetraacetic acid，EGTA)或其钠盐、二乙基三胺五乙酸(Diethyltriaminepentaacetic acid，DTPA)或其钠盐、聚磷酸盐、有机磷酸盐、磷酸酯、聚丙烯酸酯、有机磷酸盐、葡萄酸钠、或此等的混合。
在一较佳具体实施例中，本发明的一种分离脂肪间质细胞的组合物，其中该金属离子螯合剂是EDTA。
在另一较佳具体实施例中，本发明的一种分离脂肪间质细胞的组合物，其包含该第一型胶原蛋白酶2-4％(v/v)、胰蛋白酶0.1-0.3％(v/v)及EDTA0.01-0.1％(v/v)。
在一具体实施例中，本发明的一种分离脂肪间质细胞的组合物是无菌。
在一具体实施例中，本发明的一种分离脂肪间质细胞的组合物，具有使脂肪间质细胞充分游离出来，同时保护细胞不受伤害的效果。
本发明进一步提供一种分离脂肪间质细胞的方法，包括步骤：(a)取得一脂肪组织；(b)加入本发明所述分离脂肪间质细胞的组合物均质并反应后，从而获得一经消化的组织混合物；其中该组合物包含一第一型胶原蛋白酶为0.5-8％(v/v)；一胰蛋白酶为0.1-0.6％(v/v)；及一金属离子螯合剂0.01-0.2％(v/v)；(c)将步骤(b)的经消化的组织混合物离心，除去杂质，以获得含脂肪间质细胞的一滤液；(d)将一低张溶液加入步骤(c)的滤液并反应，从而获得去除血球细胞的脂肪间质细胞滤液；及(e)中和步骤(d)的滤液后离心以获得脂肪间质细胞。
在本发明的一具体实施例中，该脂肪组织是来自于一个体；其中该个体是包含动物或人类。
在本发明的一较佳具体实施例中，该金属离子螯合剂是EDTA。
在本发明的另一较佳具体实施例中，该第一型胶原蛋白酶浓度为2-4％(v/v)、胰蛋白酶浓度为0.1-0.3％(v/v)及EDTA浓度为0.01-0.1％(v/v)。
在本发明的一具体实施例中，分离脂肪间质细胞的方法是无菌。
在一较佳具体实施例中，本发明的一种分离脂肪间质细胞的方法进一步具有在总反应时间为一小时内可以充分将该脂肪组织消化，更容易大量获取脂肪间质细胞的特征。
在另一较佳具体实施例中，本发明的一种分离脂肪间质细胞的方法，其可于5克脂肪组织中分离出至少一百万颗脂肪间质细胞。
在一具体实施例中，本发明的一种分离脂肪间质细胞的方法，其中该脂肪间质细胞是可分化为脂肪细胞、造血细胞、血管内皮细胞、成骨细胞、成软骨细胞、神经细胞或上皮细胞。
在一较佳具体实施例中，本发明的一种分离脂肪间质细胞的方法，其中该脂肪间质细胞是可分化为脂肪细胞、成骨细胞或成软骨细胞。
在一较佳具体实施例中，本发明的一种分离脂肪间质细胞的方法，具有使脂肪间质细胞充分游离出来，同时保护细胞不受伤害的效果。
附图说明
图1显示本发明的一种分离脂肪间质细胞的方法的一实施步骤流程示意图。
图2显示SD大鼠大腿内侧皮下取出5公克脂肪组织，经不同浓度酵素比例配方于一小时内分离的间质细胞数量结果。
图3显示天竺鼠大腿内侧皮下取出5公克脂肪组织，经不同浓度酵素比例 配方于一小时内分离的间质细胞数量结果。
图4显示所萃取的脂肪间质细胞经筛选后的脂肪干细胞的分化能力分析。
图5显示分离的脂肪干细胞的细胞表面生物标记表现量
图6显示不同酵素配方浓度对于间质细胞存活的影响。
图7显示不同酵素种类取代本案发明组合物内容比较。
附图中符号说明：
A   0.5％第一型胶原蛋白酶+0.05％胰蛋白酶+0.02％EDTA
B   0.5％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
C   0.5％第一型胶原蛋白酶+0.2％胰蛋白酶+0.02％EDTA
D   1％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
E   2％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
F   2.5％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
G   3％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
H   4％第一型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
E’ 2％第四型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
F’ 2.5％第四型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
G’ 3％第四型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
I’ 3.5％第四型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
H’ 4％第四型胶原蛋白酶+0.1％胰蛋白酶+0.02％EDTA
具体实施方式
本发明可能以不同的形式来实施，并不仅限于下列文中所提及的实例。下列实施例仅作为本发明不同面向及特点中的代表。
以单因子变异数分析(A one-way ANOVA(analysis of variance))检定其统计上差异并利用Scheffe’s检定法进行多重比较(multiple comparisons)。显著差异的定义为显著机率值(p-value)<0.05。
实施例1快速间质细胞分离术
如图1所示，为本案发明的酵素配方运用于大鼠史－道二氏大鼠(Sprague-Dawley rat，简称SD大鼠)及天竺鼠(Genia pig)的脂肪组织的快速间质细胞分离流程示意图。
从SD大鼠及天竺鼠的大腿内侧取出5公克脂肪组织，加入等量(约5mL)的本发明的酵素配方缓冲溶液(0.5-3％(v/v)第一型胶原蛋白酶(type I collagenase)、0.1-0.3％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA溶于磷酸盐缓冲溶液(PBS)内)；置入无菌组织均质机(GentleMACs)(Miltenyi Biotec,Bergisch Gladbach,Germany)均质1分钟将组织打散成单颗细胞状态，然后浸泡于37℃水浴槽中反应10分钟；之后，再置入无菌组织均质机均质1分钟后，随后浸泡于37℃水浴槽中反应10分钟。接着，以(300×g)离心10分钟后，移除上层脂肪、结缔组织或未切碎的组织，以PBS(5mL)清洗后离心(300×g)10分钟后，加入等量(5mL)低张溶液室温静置5分钟以移除血球细胞后加入10mL PBS缓冲溶液中和反应。以3,000rpm 10分钟后移除溶液，将最下层细胞以1mL PBS重新悬浮后取样计算细胞数。总反应时间在一小时内。
实施例2间质细胞产量分析
于实施例1中所分离的脂肪间质细胞样本中取出20μL，加入20μL台酚蓝(Trypan blue)均匀混合染色后，滴入细胞计数盘中，计算存活细胞(非染色细胞)。
如图2所示，为SD大鼠大腿内侧皮下取出5公克脂肪组织，经不同浓度酵素比例配方于一小时内分离的间质细胞数量。结果显示，以3％(v/v)第一型胶原蛋白酶(type I collagenase)、0.1％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA的酵素配方(代号G)在一小时内萃取的单细胞数量最多，能分离出约9×106cells/mL颗间质细胞。
图3所示，为天竺鼠大腿内侧皮下取出5公克脂肪组织，经不同浓度酵素比例配方于一小时内分离的间质细胞数量。结果显示，以3-4％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA的酵素配方(代号G、H)在一小时内萃取的单细胞数量最多，约能分离出8×106至8.5×106cells/mL颗间质细胞。
实施例3筛选后的脂肪干细胞的分化能力
依据国际间质干细胞协会在2006年发表对间质干细胞的定义，其中间质干细胞包含三项特征：
1.细胞须能贴附于细胞培养皿；
2.表面抗原需表现CD105、CD73、或CD90,但不表现CD45、CD34、CD14或CD11b、CD79a或CD19、或HLA-DR；及
3.间质干细胞经诱导后需要具有分化成脂肪细胞，造骨细胞与软骨细胞的特性。
将实施例1中所分离的脂肪间质细胞以选择性培养基(Kerationcytr-SFM；产品编号：10724-001；GIBCO,New york,USA)(选择性留下间质干细胞，非间质干细胞则会凋亡)培养一周后可筛选出脂肪间质干细胞。如图4中的(a)中所示，所分离的脂肪间质干细胞为贴附型细胞。而利用流式细胞仪分析该分离的脂肪间质细胞亦可发现细胞表面CD271、CD73及CD90的表现量显著高于控制组值，并且CD34的表现量低于控制组值，如图5所示。故亦可得知该分离的脂肪间质细胞是属于间质干细胞。以下进一步检测该间质干细胞受诱导分化成脂肪细胞，造骨细胞与软骨细胞的能力。
前述所分离的脂肪间质干细胞经继代后，以不同诱导分化的培养基培养两周(Adipo-medium：诱发分化成脂肪细胞的培养基；Osteo-medium：诱发分化成造骨细胞的培养基；及Chondro-medium：诱发分化成软骨细胞的培养基；其中，Adipo-medium：DMEM培养基(Dulbecco’s Modified Eagle Medium)、10％胎牛血清(Fetal Bovine Serum FBS)、1％盘尼西林/链霉素(penicillin/streptomycin)、500μM 3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine IBMX)、1μM地塞米松(dexamethasone)、1μM吲哚美辛(indomethasin)、10μg/mL胰岛素(insuli)；Chondro-medium：DMEM培养基(Dulbecco’s Modified Eagle Medium)、10％胎牛血清(FBS)、1％盘尼西林/链霉素、50nM L-抗坏血酸-2-磷酸盐(L-Ascobate-2-phosphate)、6.25μg/mL胰岛素、10ng/mL TGF-β；及Osteo-medium：DMEM培养基(Dulbecco’s Modified Eagle Medium)、10％胎牛血清(FBS)、1％盘尼西林/链霉素、50μM L-抗坏血酸-2-磷酸盐(L-Ascobate-2-phosphate)、0.1μM 地塞米松、10mMβ-甘油磷酸(β-glycerophosphate)。
利用醣胺聚多醣(glycosaminoglycan，简称GAG)的测定评估间质干细胞分化成软骨细胞能力，该GAG的检测以艾尔逊蓝(Alcian blue)染色进行。在Chondro-medium两周培养之后，移除旧培养基，加入10％福尔马林(formalin)或4％聚甲醛(paraformaldehyde)固定10分钟。以蒸馏水(3mL)洗涤两次。接着以3％冰醋酸(Acetic acid)(3mL)振荡5分钟。移除全部的液体后，于盘中加入艾尔逊蓝(1％)，振荡15分钟，移除染剂后以蒸馏水(3mL)洗涤2-3次后进行拍照记录。
如图4的(b)所示，艾尔逊蓝对于细胞外基质GAG进行染色，该GAG染色呈蓝色且细胞聚集，为软骨细胞的特性。
利用油红O(Oil Red O)对脂肪细胞中油滴的染色，评估间质干细胞分化成脂肪细胞的能力。在Adipo-medium培养两周之后，移除旧培养基，加入10％福尔马林或4％聚甲醛固定10分钟。以蒸馏水(3mL)洗涤两次。移除全部的液体后，于盘中加入Oil Red O(0.5％)，摆荡10分钟，移除染剂后以蒸馏水(3mL)洗涤2-3次，加入蒸馏水后进行拍照记录。
如图4的(c)所示，该染色细胞内油滴呈红色点状，为脂肪细胞的特性。
利用茜红(Alizarin-red)染色检测所分离的脂肪间质干细胞诱发骨生成作用的效果。在Osteo-medium培养两周之后，移除旧培养基，加入4％福尔马林固定15分钟。以蒸馏水(3mL)洗涤两次。移除全部的液体后，于盘中加入2％茜红溶液，以常温下反应20分，移除染剂后以蒸馏水(3mL)洗涤2-3次后进行拍照记录。
如图4的(d)所示，该染色细胞产生的钙沉积呈红色点状，为造骨细胞的特性。
实施例4不同酵素配方浓度对于间质细胞存活的影响
经实施例1所分离的脂肪间质细胞以选择性培养基培养(Kerationcytr-SFM；产品编号：10724-001；GIBCO)(选择性留下间质干细胞，非间质干细胞则会凋亡)培养一周后可筛选出脂肪间质干细胞。以下述三种在实施例1所揭示的较佳脂肪间质细胞分离产量的酵素配方进行细胞毒性试验：
1. 2.5％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA的酵素配方；
2. 3％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA的酵素配方；及
3. 4％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶(Trypsin)及0.02％(v/v)EDTA的酵素配方。
将105颗脂肪间质干细胞置于该等酵素配方，并培养0.5、1、2小时。于培养0.5、1、2小时后，取出细胞进行实施例2的Trypan-blue染色进行细胞存活率的计数，如图6所示，该等配方并未有明显细胞毒杀现象。
实施例5不同酵素种类取代比较
将相同配方浓度更换成类似本发明中使用的不同型酵素：2％(v/v)第四型胶原蛋白酶、0.1％(v/v)胰蛋白酶(Trypsin)、0.02％(v/v)EDTA；2.5％第四型胶原蛋白酶、0.1％Trypsin、0.02％EDTA；3％第四型胶原蛋白酶、0.1％Trypsin、0.02％EDTA；3.5％第四型胶原蛋白酶、0.1％Trypsin、 0.02％EDTA；4％第四型胶原蛋白酶、0.1％Trypsin、0.02％EDTA，并依实施例1所教示的分离脂肪间质细胞操作方法进行实施。其结果如图7所示，本实施例的配方并无法如同3％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶及0.02％(v/v)EDTA的酵素配方得到相近的细胞数。因此3％(v/v)第一型胶原蛋白酶、0.1％(v/v)胰蛋白酶及0.02％(v/v)EDTA的酵素配方在快速分离脂肪间质细胞中是无可取代的。
以上内容阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其它不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受前面公开的具体实施例的限制。

Claims (9)

  1. 一种分离脂肪间质细胞的组合物，其包含一0.5-8％(v/v)的第一型胶原蛋白酶；一0.1-0.6％(v/v)的胰蛋白酶；及一0.01-0.2％(v/v)的金属离子螯合剂。
  2. 根据权利要求1所述的组合物，其中该金属离子螯合剂选自乙烯二胺四乙酸或其钠盐、乙二醇双氨乙基醚四乙酸或其钠盐、二乙基三胺五乙酸或其钠盐、聚磷酸盐、有机磷酸盐、磷酸酯、聚丙烯酸酯、有机磷酸盐、葡萄酸钠、或此等的混合。
  3. 根据权利要求2所述的组合物，其中该金属离子螯合剂是EDTA。
  4. 一种分离脂肪间质细胞的方法，包括步骤：
    (a)取得一脂肪组织；
    (b)加入权利要求1所述的组合物均质并反应后，从而获得一经消化的组织混合物；其中该组合物包含一0.5-8％(v/v)的第一型胶原蛋白酶；一0.1-0.6％(v/v)的胰蛋白酶；及一0.01-0.2％(v/v)的金属离子螯合剂；
    (c)将步骤(b)的经消化的组织混合物离心，除去杂质，以获得含脂肪间质细胞的一第一滤液；
    (d)将一低张溶液加入步骤(c)的第一滤液，从而获得去除血球细胞的脂肪间质细胞的一第二滤液；及
    (e)中和步骤(d)的第二滤液并离心。
  5. 根据权利要求4所述的方法，其中该金属离子螯合剂选自乙烯二胺四乙酸或其钠盐、乙二醇双氨乙基醚四乙酸或其钠盐、二乙基三胺五乙酸或 其钠盐、聚磷酸盐、有机磷酸盐、磷酸酯、聚丙烯酸酯、有机磷酸盐、葡萄酸钠、或此等的混合。
  6. 根据权利要求5所述的方法，其中该金属离子螯合剂系EDTA。
  7. 根据权利要求4所述的方法，其进一步具有在总反应时间为一小时内将一脂肪组织消化，以获取该脂肪间质细胞的特征。
  8. 根据权利要求4所述的方法，其具有于5克脂肪组织中分离出至少一百万颗脂肪间质细胞的功效。
  9. 根据权利要求4所述的方法，其中该脂肪间质细胞是可分化为脂肪细胞、造血细胞、血管内皮细胞、成骨细胞、成软骨细胞、神经细胞或上皮细胞。
PCT/CN2015/091449 2015-10-08 2015-10-08 一种快速分离脂肪间质细胞的组合物 WO2017059566A1 (zh)

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