不同非接触式超声波处理获得脂肪源基质血管组分细胞方法的比较
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摘要
背景:探索能够高效、稳定获取基质血管组分细胞的方法对于临床应用有非常重要的价值。目前分离基质血管组分的方法主要包括酶解法、推注破碎法、接触式超声波破碎法等,存在安全性和细胞存活率低、材料污染等不足,而非接触式超声波破碎法则能很好的解决这个问题。目的:寻找一种最优的非接触式超声波处理获得脂肪源基质血管组分细胞的方法。方法:实验所用人体脂肪组织(皮下脂肪)由广州中家医家庭医生整形美容医院提供,体检无其他基础疾病,取材前均获知情同意并签署知情同意书。以3种不同的超声波仪器(超声波清洗仪功率约为800 W、超声波细胞破碎仪功率约为1 000 W、非接触式超声波细胞破碎仪功率约为1 200 W)对脂肪材料进行非接触式超声处理15,20,25 min,以接触式超声波细胞破碎法为对照,功率为100 W,超声处理16 s,分别检测基质血管组分细胞大小、存活数量、细胞活力、细胞碎片率和微生物感染度。该研究实施符合广州中家医家庭医生整形美容医院的相关伦理要求。结果与结论:(1)3种方法所获得的基质血管组分细胞活性均高于接触式超声波破碎法(P <0.05);(2)3种方法所获得的基质血管组分细胞污染度均低于接触式超声波破碎法;(3)3种方法获得的基质血管组分细胞大小差异无显著性意义(P>0.05);(4)3种方法所获得的基质血管组分细胞碎片均少于接触式超声波破碎法(P<0.05),其中非接触式超声波细胞破碎仪处理20 min后采用酶解方法获取基质血管组分细胞的效果最好。
BACKGROUND: It is of great clinical significance to explore methods for efficient and stable access to stromalvascular component cells. Current methods for separating stromal vascular components mainly include enzymatic hydrolysis, bolus injection method, and contact ultrasonic pulverization method. For contact ultrasonic pulverization, there are many problems to be solved, such as low safety, low cell survival rate, and material contamination. Therefore, non-contact ultrasonic pulverization can solve these problems well. OBJECTIVE: To find an optimal method for harvesting adipose-derived stromal vascular fraction cells by non-contact ultrasound approach. METHODS: The human body fat tissue(subcutaneous fat) used was provided by the Plastic Surgery Hospital of Chinese Family Physician. Patients were eligible if they had no other basic diseases in the physical examination. All the patients were informed of study protocol and signed informed consent before sampling. The fat tissues were treated with three different ultrasonic instruments(approximately 800 W for ultrasonic cleaner, 1 000 W for ultrasonic cell crusher, and 1 200 W for non-contact ultrasonic cell crusher) for 15, 20, and 25 minutes. Contact ultrasonic crushing method(100 W, 16 seconds) was used as control. Stromal vascular fraction cell size, number of survived cells, cell viability, cell fragmentation rate and microbial infection were measured. The study was implemented in accordance with the relevant ethical requirements of Plastic Surgery Hospital of Chinese Family Physician. RESULTS AND CONCLUSION: The viability of stromal vascular fraction cells obtained by the three methods was significantly higher than that by the contact ultrasonic crushing method(P < 0.05). The contamination degree of stromal vascular fraction cells obtained by the three methods was significantly lower than that obtained by the contact ultrasonic crushing method(P < 0.05). There was no significant difference in the size of stromal vascular fraction cells obtained by the three methods(P > 0.05). The cell fragments of stromal vascular fraction cells obtained by the three methods were significantly less than those by contact ultrasonic crushing method(P < 0.05). It is preferred to perform 20 minutes treatment by non-contact ultrasonic cell crusher followed by enzymatic separation.
BACKGROUND: It is of great clinical significance to explore methods for efficient and stable access to stromalvascular component cells. Current methods for separating stromal vascular components mainly include enzymatic hydrolysis, bolus injection method, and contact ultrasonic pulverization method. For contact ultrasonic pulverization, there are many problems to be solved, such as low safety, low cell survival rate, and material contamination. Therefore, non-contact ultrasonic pulverization can solve these problems well. OBJECTIVE: To find an optimal method for harvesting adipose-derived stromal vascular fraction cells by non-contact ultrasound approach. METHODS: The human body fat tissue(subcutaneous fat) used was provided by the Plastic Surgery Hospital of Chinese Family Physician. Patients were eligible if they had no other basic diseases in the physical examination. All the patients were informed of study protocol and signed informed consent before sampling. The fat tissues were treated with three different ultrasonic instruments(approximately 800 W for ultrasonic cleaner, 1 000 W for ultrasonic cell crusher, and 1 200 W for non-contact ultrasonic cell crusher) for 15, 20, and 25 minutes. Contact ultrasonic crushing method(100 W, 16 seconds) was used as control. Stromal vascular fraction cell size, number of survived cells, cell viability, cell fragmentation rate and microbial infection were measured. The study was implemented in accordance with the relevant ethical requirements of Plastic Surgery Hospital of Chinese Family Physician. RESULTS AND CONCLUSION: The viability of stromal vascular fraction cells obtained by the three methods was significantly higher than that by the contact ultrasonic crushing method(P < 0.05). The contamination degree of stromal vascular fraction cells obtained by the three methods was significantly lower than that obtained by the contact ultrasonic crushing method(P < 0.05). There was no significant difference in the size of stromal vascular fraction cells obtained by the three methods(P > 0.05). The cell fragments of stromal vascular fraction cells obtained by the three methods were significantly less than those by contact ultrasonic crushing method(P < 0.05). It is preferred to perform 20 minutes treatment by non-contact ultrasonic cell crusher followed by enzymatic separation.
引文
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[19]Ferrario M,Guerrero S.Impact of a combined processing technology involving ultrasound and pulsed light on structural and physiological changes of Saccharomyces cerevisiae KE 162 in apple juice.Food Microbiol.2017;65:83-94.
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[24]Micu R,Chicea AL,Bratu DG,et al.Ultrasound and magnetic resonance imaging in the prenatal diagnosis of open spina bifida.Med Ultrason.2018;20(2):221-227.
[25]Cen LP,Ng TK.Stem cell therapy for retinal ganglion cell degeneration.Neural Regen Res.2018;13(8):1352-1353.
[26]龚军辉,王晶.稀释涂布平板法计数活菌的方法简介[J].生物学教学,2018,43(2):70-71.
[27]Majka SM,Miller HL,Helm KM,et al.Analysis and isolation of adipocytes by flow cytometry.Methods Enzymol.2014;537:281-296.
[28]Boumelhem BB,Assinder SJ,Bell-Anderson KS,et al.Flow cytometric single cell analysis reveals heterogeneity between adipose depots.Adipocyte.2017;6(2):112-123.
[29]马晨光,薛迪.干细胞研究与应用的伦理问题、伦理规范与伦理实证研究[J].中国医学伦理学,2019,32(1):26-29.
[30]Pierantozzi E,Badin M,Vezzani B,et al.Human pericytes isolated from adipose tissue have better differentiation abilities than their mesenchymal stem cell counterparts.Cell Tissue Res.2015;361(3):769-778.
[31]Fan J,Sun Z.The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells.Stem Cells.2016;34(6):1615-1625.
[32]王延光.人类胚胎干细胞的来源与伦理思考[J].医学与哲学,2002,23(2):7-10.
[33]Sterodimas A,de Faria J,Nicaretta B,et al.Tissue engineering with adipose-derived stem cells(ADSCs):current and future applications.JPlast Reconstr Aesthet Surg.2010;63(11):1886-1892.
[34]Roxburgh J,Metcalfe AD,Martin YH.The effect of medium selection on adipose-derived stem cell expansion and differentiation:implications for application in regenerative medicine.Cytotechnology.2016;68(4):957-967.
[35]Chen D,Liu S,Ma H,et al.Paracrine factors from adiposemesenchymal stem cells enhance metastatic capacity through Wnt signaling pathway in a colon cancer cell co-culture model.Cancer Cell Int.2015;15:42.
[36]Sun M,He Y,Zhou T,et al.Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model.Biomed Res Int.2017;2017:3105780.
[37]Schendel SA.Autologous Adipose-Derived Tissue Matrix Part I:Biologic Characteristics.Aesthet Surg J.2017;37(9):1062-1068.
[38]Priglinger E,Schuh CMAP,Steffenhagen C,et al.Improvement of adipose tissue-derived cells by low-energy extracorporeal shock wave therapy.Cytotherapy.2017;19(9):1079-1095.
[2]Heo JS,Choi Y,Kim HS,et al.Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow,umbilical cord blood,placenta and adipose tissue.Int J Mol Med.2016;37(1):115-125.
[3]Xiong BJ,Tan QW,Chen YJ,et al.The Effects of Platelet-Rich Plasma and Adipose-Derived Stem Cells on Neovascularization and Fat Graft Survival.Aesthetic Plast Surg.2018;42(1):1-8.
[4]Zhang Y,Cai J,Zhou T,et al.Improved Long-Term Volume Retention of Stromal Vascular Fraction Gel Grafting with Enhanced Angiogenesis and Adipogenesis.Plast Reconstr Surg.2018;141(5):676e-686e.
[5]Rodbell M.Metabolism of isolated fat cells.i.effects of hormones on glucose metabolism and lipolysis.J Biol Chem.1964;239:375-380.
[6]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.
[7]Gaiba S,Fran?a LP,Fran?a JP,et al.Characterization of human adipose-derived stem cells.Acta Cir Bras.2012;27(7):471-476.
[8]Yao Y,Dong Z,Liao Y,et al.Adipose Extracellular Matrix/Stromal Vascular Fraction Gel:A Novel Adipose Tissue-Derived Injectable for Stem Cell Therapy.Plast Reconstr Surg.2017;139(4):867-879.
[9]Garza RM,Rennert RC,Paik KJ,et al.Studies in fat grafting:Part IV.Adipose-derived stromal cell gene expression in cell-assisted lipotransfer.Plast Reconstr Surg.2015;135(4):1045-1055.
[10]Paik KJ,Zielins ER,Atashroo DA,et al.Studies in Fat Grafting:Part V.Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent.Plast Reconstr Surg.2015;136(1):67-75.
[11]Gimble JM,Katz AJ,Bunnell BA.Adipose-derived stem cells for regenerative medicine.Circ Res.2007;100(9):1249-1260.
[12]Nie C,Yang D,Xu J,et al.Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis.Cell Transplant.2011;20(2):205-216.
[13]Shah FS,Wu X,Dietrich M,et al.A non-enzymatic method for isolating human adipose tissue-derived stromal stem cells.Cytotherapy.2013;15(8):979-985.
[14]刘洋,潘磊,金永利.鼠脂肪干细胞体外培养及软骨分化的研究[J].临床医药文献电子杂志,2018,5(86):188.
[15]Tonnard P,Verpaele A,Peeters G,et al.Nanofat grafting:basic research and clinical applications.Plast Reconstr Surg.2013;132(4):1017-1026.
[16]Premaratne GU,Ma LP,Fujita M,et al.Stromal vascular fraction transplantation as an alternative therapy for ischemic heart failure:anti-inflammatory role.J Cardiothorac Surg.2011;6:43.
[17]Brennen CE.Cavitation and Bubble Dynamics.Oxford:Oxford University Press,1995.
[18]Zhu J,Wang Y,Li X,et al.Combined effect of ultrasound,heat,and pressure on Escherichia coli O157:H7,polyphenol oxidase activity,and anthocyanins in blueberry(Vaccinium corymbosum)juice.Ultrason Sonochem.2017;37:251-259.
[19]Ferrario M,Guerrero S.Impact of a combined processing technology involving ultrasound and pulsed light on structural and physiological changes of Saccharomyces cerevisiae KE 162 in apple juice.Food Microbiol.2017;65:83-94.
[20]Yang YH,Lin SY.Medical ultrasonic treatment and its applications in medicine[C].上海:2012全国压电和声波理论及器件技术研讨会论文集,2012.
[21]Leonov GV,Khmelev VN,Barsukov RV,et al.Increasing of ultrasonic technologies efficiency and development of ultrasonic devices for industry,medicine and agriculture.International Siberian Workshop on Electron Devices and Materials,2004.
[22]Kokhuis T,Skachkov I,Naaijkens B,et al.Characterization of microbubble-loaded stem cells for targeted cell therapy.IEEEInternational Ultrasonics Symposium,2013:1146-1149.
[23]Garbern JC,Lee RT.Cardiac stem cell therapy and the promise of heart regeneration.Cell Stem Cell.2013;12(6):689-698.
[24]Micu R,Chicea AL,Bratu DG,et al.Ultrasound and magnetic resonance imaging in the prenatal diagnosis of open spina bifida.Med Ultrason.2018;20(2):221-227.
[25]Cen LP,Ng TK.Stem cell therapy for retinal ganglion cell degeneration.Neural Regen Res.2018;13(8):1352-1353.
[26]龚军辉,王晶.稀释涂布平板法计数活菌的方法简介[J].生物学教学,2018,43(2):70-71.
[27]Majka SM,Miller HL,Helm KM,et al.Analysis and isolation of adipocytes by flow cytometry.Methods Enzymol.2014;537:281-296.
[28]Boumelhem BB,Assinder SJ,Bell-Anderson KS,et al.Flow cytometric single cell analysis reveals heterogeneity between adipose depots.Adipocyte.2017;6(2):112-123.
[29]马晨光,薛迪.干细胞研究与应用的伦理问题、伦理规范与伦理实证研究[J].中国医学伦理学,2019,32(1):26-29.
[30]Pierantozzi E,Badin M,Vezzani B,et al.Human pericytes isolated from adipose tissue have better differentiation abilities than their mesenchymal stem cell counterparts.Cell Tissue Res.2015;361(3):769-778.
[31]Fan J,Sun Z.The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells.Stem Cells.2016;34(6):1615-1625.
[32]王延光.人类胚胎干细胞的来源与伦理思考[J].医学与哲学,2002,23(2):7-10.
[33]Sterodimas A,de Faria J,Nicaretta B,et al.Tissue engineering with adipose-derived stem cells(ADSCs):current and future applications.JPlast Reconstr Aesthet Surg.2010;63(11):1886-1892.
[34]Roxburgh J,Metcalfe AD,Martin YH.The effect of medium selection on adipose-derived stem cell expansion and differentiation:implications for application in regenerative medicine.Cytotechnology.2016;68(4):957-967.
[35]Chen D,Liu S,Ma H,et al.Paracrine factors from adiposemesenchymal stem cells enhance metastatic capacity through Wnt signaling pathway in a colon cancer cell co-culture model.Cancer Cell Int.2015;15:42.
[36]Sun M,He Y,Zhou T,et al.Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model.Biomed Res Int.2017;2017:3105780.
[37]Schendel SA.Autologous Adipose-Derived Tissue Matrix Part I:Biologic Characteristics.Aesthet Surg J.2017;37(9):1062-1068.
[38]Priglinger E,Schuh CMAP,Steffenhagen C,et al.Improvement of adipose tissue-derived cells by low-energy extracorporeal shock wave therapy.Cytotherapy.2017;19(9):1079-1095.