Corning® CellBIND®表面培养皿促进脐带源间充质干细胞生长

doi:10.3969/j.issn.2095-4344.2014.10.011

摘要/Abstract

摘要：

背景：培养体系微环境影响干细胞体外扩增，寻找有效的促进细胞贴壁和增殖的培养方法尤为重要。
目的：比较不同细胞培养材质对细胞体外扩增的影响。
方法：将5.0×10⁴培养的人脐带源间充质干细胞分别接种于包被/不包被鸟氨酸Corning^®聚苯乙烯培养皿或Corning^® CellBIND^®表面培养皿中培养，分别观察细胞的贴壁、增殖状态、与细胞黏附和细胞增殖有关的蛋白的表达及细胞标志物的表达。
结果与结论：与其他类型的培养皿相比，包被多聚鸟氨酸的Corning^® CellBIND^®表面培养皿在24 h内能够促进人脐带源间充质干细胞贴壁，同时也能使培养的人脐带源间充质干细胞较早的进入对数生长期，细胞增殖数量相对较多，虽然对人脐带源间充质干细胞表面标志物CD73，CD90和CD105的表达没有影响，但能促进细胞中黏附蛋白的表达。且Corning® CellBIND^®表面培养皿较Corning^®聚苯乙烯培养皿更能促进人脐带源间充质干细胞的贴壁和增殖，同时也对细胞表型的表达无影响。提示Corning^®CellBIND^®表面培养皿能够促进细胞吸附，增加细胞数量，且对细胞周期蛋白以及细胞表型的表达没有影响，且包被多聚鸟氨酸能够进一步促进细胞的贴壁和增殖，并稳定表达人脐带源间充质干细胞的细胞表型。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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关键词: 干细胞, 脐带脐血干细胞, 干细胞基础实验, Corning^®, CellBIND^®表面培养皿, 细胞贴壁, 细胞增殖, 细胞表型, 脐带源间充质干细胞, 多聚鸟氨酸

Abstract:

BACKGROUND: Stem cells expansion technology in vitro is affected by the microenvironment of the culture system. So, to find an effective method is particularly important to promote cell adherent and growth.
OBJECTIVE: To compare the effects of different culture media on cell expansion.
METHODS: Human umbilical cord mesenchymal stem cells at a density of 5.0×10⁴ cells/cm² were inoculated into Corning^®polystyrene culture dishes coated with or without poly-L-ornithine and Corning^® CellBIND culture dishes. Cell adhesion and proliferation were observed, and expressions of cell adhesion proteins and cell markers were detected.
RESULTS AND CONCLUSION: Cell adhesion was promoted when cells were cultured in Corning^® CellBIND® Surface medium coated with poly-L-ornithine for 24 hours, and the cultured cells grew at the logarithmic phase. The cell proliferation was also enhanced, and the cells expressed cell adhesion protein but not the cell markers of CD73, CD90, CD105. Corning^®CellBIND^® Surface medium was superior to Corning^®polystyrene culture medium in the improvement of cell adhesion and proliferation. Additionally, both of these two media showed no influence on cell phenotype. These findings indicate that Corning^®CellBIND^® Surface medium can promote cell adhesion and proliferation, but shows no effects on cyclin and cell phenotype. This medium coated with poly-L-ornithine can further accelerate cell adhesion and proliferation, and stably express cell phenotype of human umbilical cord mesenchymal stem cells.

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Key words: mesenchymal stem cells, cell culture techniques, cell proliferation, ornithin

中图分类号:

R394.2

宛莹，贲亮，官子楸，李超，张世冬，聂德志. Corning^®CellBIND^®表面培养皿促进脐带源间充质干细胞生长[J]. 中国组织工程研究, 2014, 18(10): 1547-1553.

Wan Ying, Ben Liang, Guan Zi-qiu, Li Chao, Zhang Shi-dong, Nie De-zhi. Effects of Corning® CellBIND® Surface medium on growth of human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(10): 1547-1553.

图/表（结果） 5

Effects of Corning^® CellBIND^® Surface dish coated with poly-L-ornithine on cell adhesion Cell numbers and morphology were observed in different culture dishes within 24 hours as shown in Figure 1. Cells cultured in the Corning^® culture dish were most adhered and spindle-shaped, resembling fibroblasts. Corning^® CellBIND^® Surface culture dish also exhibited the powerful effect on cell growth^[15-22], and the number of adherent cells was increased with the time prolonged within 24 hours. Depending on the treatment of microwave plasma process for treating the culture surface, Corning^® CellBIND^® Surface culture dish coated with poly-L-ornithine exhibited a better effect on cell attachment than Corning^® tissue culture polystyrene dish coated with poly-L-ornithine (Figure 2).

Effects of Corning^® CellBIND^® Surface dish coated with poly-L-ornithine on cell proliferation

Cells proliferating in the different culture surfaces were observed in 7 days as shown in Figure 3. The results showed the growth curve of hUMSCs presented inverted “S”, and cells exhibited logarithmic growth on days 3, 4, 5 and 6. Owing to the effect of poly-L-ornithine on cell adhesion, Corning^® tissue culture polystyrene dish and Corning^® CellBIND^® Surface dish coated with poly-L-ornithine improved cell growth at 7 days compared with the culture dishes that coated without poly-L-ornithine. However, Corning^® CellBIND^® Surface dish exerted the better effect on cell proliferation under any treatment on the surface of dish compared with Corning^® tissue culture polystyrene dish (Figure 3).

Effects of Corning^® CellBIND^® Surface dish coated with poly-L-ornithine on cell adhesion and cell proliferative protein expressions

To test the effect of Corning^®CellBIND^® Surface culture dish on cell attachment and cell proliferative protein expressions, first, we detected cell proliferative protein including cyclinA, cyclinE and PCNA expressions. The results showed that neither Corning^® CellBIND^® Surface culture dish, Corning^®tissue culture polystyrene dish nor those coated with poly-L-ornithine influence the expressions of PCNA, cyclinA or cyclinE by western blot analysis (P > 0.05). Furthermore, the expressions of adhesion cytokines were examined and the expression of fibronectin was significant increased in Corning^® tissue culture polystyrene dish coated with poly-L-ornithine and Corning^® CellBIND^® Surface culture dish coated with or without poly-L-ornithine (P < 0.05) (Figure 4).

Effects of Corning^® CellBIND^® Surface dish coated with poly-L-ornithine on expression of cell surface markers

We previously demonstrated that similarly processed hUMSCs expressed high levels of matrix receptors (CD73, CD90 and CD105) as assessed by flow cytometry. In addition, all adherent hUMSCs did not express CD45, CD34, CD14, CD19 or HLA-DR (data was not shown). In order to determine the effect of Corning^® CellBIND^® Surface culture dish on hUMSCs phenotype, we examined the levels of CD73, CD90 and CD105 in the four different groups. As our predicted, hUMSCs highly expressed CD73, CD90 and CD105 in the four groups as same as our isolated cells from human umbilical cords before. It implied that Corning^® CellBIND^® Surface culture dish coated with or without poly-L-ornithine did not affect the cell phenotype (Figure 5).

参考文献

[1]Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant. 2011;20(1):5-14.

[2]Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J (Engl). 2005;118(23):1987-1993.

[3]Liu S, Yuan M, Hou K, et al. Immune characterization of mesenchymal stem cells in human umbilical cord Wharton's jelly and derived cartilage cells. Cell Immunol. 2012; 278(1-2):35-44.

[4]Song WW, Bai H, Wang CB, et al. Effects of hypoxia on the proliferation of human bone marrow mesenchymal stem cells. Zhonghua Yi Xue Za Zhi. 2010;90(30):2149- 2152.

[5]Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells. 2003; 21(1):50-60.

[6]Poulsson AH, Mitchell SA, Davidson MR, et al. Attachment of human primary osteoblast cells to modified polyethylene surfaces. Langmuir. 2009;25(6):3718-3727.

[7]Shen M, Horbett TA. The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces. J Biomed Mater Res. 2001;57(3):336-345.

[8]Pon-On W, Charoenphandhu N, Teerapornpuntakit J, et al. In vitro study of vancomycin release and osteoblast-like cell growth on structured calcium phosphate-collagen. Mater Sci Eng C Mater Biol Appl. 2013;33(3):1423-1431.

[9]Rumian L, Wojak I, Scharnweber D, et al. Resorbable scaffolds modified with collagen type I or hydroxyapatite: in vitro studies on human mesenchymal stem cells. Acta Bioeng Biomech. 2013;15(1):61-67.

[10]Gotman I, Ben-David D, Unger RE, et al. Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds. Acta Biomater. 2013;9(9): 8440-8448.

[11]Long X, Matsumoto R, Yang P, et al. Effect of human mesenchymal stem cells on the growth of HepG2 and Hela cells. Cell Struct Funct. 2013;38(1):109-121.

[12]Dolley-Sonneville PJ, Romeo LE, Melkoumian ZK. Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions. PLoS One. 2013;8(8):e70263.

[13]Estrada JC, Torres Y, Benguría A, et al. Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death Dis. 2013;4:e691.

[14]Capra E, Beretta R, Parazzi V, et al. Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages. Proteome Sci. 2012;10(1):46.

[15]Harnett EM, Alderman J, Wood T. The surface energy of various biomaterials coated with adhesion molecules used in cell culture. Colloids Surf B Biointerfaces. 2007;55(1): 90-97.

[16]Favi PM, Benson RS, Neilsen NR, et al. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds. Mater Sci Eng C Mater Biol Appl. 2013;33(4):1935-1944.

[17]Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci. 2008;121(Pt 3):255-264.

[18]Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007;8(3):221-233.

[19]van Kooten TG, Spijker HT, Busscher HJ. Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions. Biomaterials. 2004;25(10): 1735-1747.

[20]Spoerke ED, Stupp SI. Synthesis of a poly(L-lysine)-calcium phosphate hybrid on titanium surfaces for enhanced bioactivity. Biomaterials. 2005; 26(25):5120-5129.

[21]Lu H, Guo L, Kawazoe N, et al. Effects of poly(L-lysine), poly(acrylic acid) and poly(ethylene glycol) on the adhesion, proliferation and chondrogenic differentiation of human mesenchymal stem cells. J Biomater Sci Polym Ed. 2009; 20(5-6):577-589.

[22]Steele JG, Dalton BA, Johnson G, et al. Polystyrene chemistry affects vitronectin activity: an explanation for cell attachment to tissue culture polystyrene but not to unmodified polystyrene. J Biomed Mater Res. 1993;27(7): 927-940.

[23]Xu Y, Xiao Q, Tian Y, et al. Biological effects of the extracellular matrix on rat bone marrow mesenchymal stem cells. US Chin J Lymphology Oncol. 2007;10(1):26-28.

[24]Shadpour H, Sims CE, Thresher RJ, et al. Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays. Cytometry A. 2009;75(2):121-129.

[25]Wang Y, Young G, Bachman M, et al. Collection and expansion of single cells and colonies released from a micropallet array. Anal Chem. 2007;79(6):2359-2366.

[26]Xu W, Luikart AM, Sims CE, et al. Contact printing of arrayed microstructures. Anal Bioanal Chem. 2010;397(8): 3377-3385.

[27]Gunn NM, Bachman M, Li GP, et al. Fabrication and biological evaluation of uniform extracellular matrix coatings on discontinuous photolithography generated micropallet arrays. J Biomed Mater Res A. 2010;95(2):401-412.

[28]Shadpour H, Sims CE, Allbritton NL. Enrichment and expansion of cells using antibody-coated micropallet arrays. Cytometry A. 2009;75(7):609-618.

[29]Beaulieu I, Geissler M, Mauzeroll J. Oxygen plasma treatment of polystyrene and Zeonor: substrates for adhesion of patterned cells. Langmuir. 2009;25(12): 7169-7176.

[30]Kleinhans C, Barz J, Wurster S, et al. Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells. Biotechnol J. 2013;8(3):327-337.

[31]Lee JH, Jung HW, Kang IK, et al. Cell behaviour on polymer surfaces with different functional groups. Biomaterials. 1994;15(9):705-711.

[32]Jankowski RJ, Haluszczak C, Trucco M, et al. Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther. 2001;12(6):619-628.

[33]Yun JK, DeFife K, Colton E, et al. Human monocyte/macrophage adhesion and cytokine production on surface-modified poly(tetrafluoroethylene/ hexafluoropropylene) polymers with and without protein preadsorption. J Biomed Mater Res. 1995;29(2):257-268.

[34]DeFife KM, Yun JK, Azeez A, et al. Adhesion and cytokine production by monocytes on poly(2-methacryloyloxyethyl phosphorylcholine-co-alkyl methacrylate)-coated polymers. J Biomed Mater Res. 1995;29(4):431-439.

[35]Lee JH, Lee HB. A wettability gradient as a tool to study protein adsorption and cell adhesion on polymer surfaces. J Biomater Sci Polym Ed. 1993;4(5):467-481.

[36]Fisher RP. The CDK network: linking cycles of cell division and gene expression. Genes Cancer. 2012;3(11-12):731- 738.

[37]Opas M, Dziak E. Adhesion, spreading, and proliferation of cells on protein carpets: effects of stability of a carpet. In Vitro Cell Dev Biol. 1991;27A(11):878-885.

[38]Jenney CR, Anderson JM. Adsorbed serum proteins responsible for surface dependent human macrophage behavior. J Biomed Mater Res. 2000;49(4):435-447.

[39]Flanagan LA, Rebaza LM, Derzic S, et al. Regulation of human neural precursor cells by laminin and integrins. J Neurosci Res. 2006;83(5):845-856.

引言

材料方法

Design

Cytology in vitro study.

Time and setting

The study was completed at Jilin Province Key Laboratory of Tissue Engineering Research from October 2012 to May 2013.

Materials

Cells

hUMSCs were donated with the consent of the healthy patients and approval of the Research Ethics Committee in Siping

Central Hospital (Jilin, China). hUMSCs were harvested from fresh human umbilical cords using plastic adherence method, and then cells expanded in culture flasks.

Culture dish

Corning^® tissue culture polystyrene dish and Corning^® CellBIND^® Surface culture dish were produced by Corning, New York, USA. According to the following procedures: the 0.01% poly-L-ornithine solution diluted 10 times with sterilized injection water. The dishes were coated with

5 mL of the diluted poly-L-ornithine solution at 37 ℃ for

2 hours, and after solution removal, the dishes were aired in the biosafe cabinet^[9-11]. For Corning^®CellBIND^® technology, a microwave plasma process is used for treating the culture surface. This process improves cell attachment by incorporating significantly more oxygen into the cell culture surface, rendering the hydrophilicity (wettable) and increasing surface stability.

Other reagents

Methods

hUMSCs isolation and culture

hUMSCs were detached from culture flasks using Trypsin/EDTA seeded to the wells at a density of 5.0 × 10⁴ cells/cm² and cultured at 37 ℃ in 5% CO₂. At culture termination, the cells were washed once with PBS before further processing as described in the respective method.

Cell grouping

There were four groups, namely Corning^® polystyrene culture dishes, Corning^® polystyrene culture dishes coated with poly-L-ornithine; Corning^®CellBIND^® Surface culture dish and Corning^® CellBIND^® Surface culture dish coated poly-L-ornithine. The changes in cell proliferative protein and adhesion protein expressions, cell density, cell proliferation and cell morphology were detected.

MSCs attachment and proliferation detected by microscope

MSCs (5.0×10⁴) were plated onto 60 mm Corning^® tissue culture polystyrene dish and Corning^®CellBIND^® Surface dish coated with or without poly-L-ornithine. Triplicate dishes were collected by trypsinization at the indicated periods of time and the cells were counted using Olympus CKX41SF microscope.

Expression of cell surface markers detected by flow cytometry

hUMSCs were characterized using flow cytometry (FACSCalibur, Becton, Dickinson and Company, NJ, USA), and were incubated with the following anti-human primary antibodies: CD73-FITC conjugated; CD90-FITC conjugated; CD105-PE conjugated; unconjugated markers were reacted with anti-mouse PE-conjugated secondary antibody. A total of 500 000 labeled cells were analyzed and the data obtained were analyzed using BD CellQuest software^[12-14].

The expressions of cell adhesion and cell proliferative proteins detected by western blot assay

To verify that the adhesion proteins are adsorbed to the cell culture dishes coated with or without poly-L-ornithine, and the changes of hUMSCs proliferative protein expression in different culture dishes, fibronectin, cyclinA, cyclinE and PCNA expressions were detected by western blot analysis. The cells were harvested and washed with cold PBS. Then 30-50 μg of cellular or nuclear extract was subjected to SDS-PAGE in 10%-15% gradient gels. Proteins in the gel were transferred onto polyvinylidene fluoride membranes and probed with fibronectin, cyclinA, cyclinE and PCNA antibodies in 5% skim milk PBS containing 0.1% Tween-20 (PBST) for 24 hours at 4 ℃. The blots were then incubated with 1:2 000 dilutions of horseradish peroxidase-conjugated goat anti-rabbit or horseradish peroxidase-conjugated goat anti-mouse IgG for 1 hour at room temperature. The membrane was washed extensively before detection using the chemiluminescence BeyoECL plus kit (Thermo Fisher, USA). The membranes were then stripped and reprobed with β-actin antibody as the loading control. Judgment based on the band depths of color was conducted to analyze the expression of the primary antibody.

Main outcome measures

(1) The effect of different culture dishes coated with poly-L-ornithine on cell adhesion protein, cell proliferation, cell cyclin expressions; (2) the effect of different culture dishes coated with poly-L-ornithine on cell phenotype expressions.

Statistical analysis

The data were expressed as mean±SD and statistically processed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. Calculations were performed using the GraphPad Prism 5.0 program (GraphPad Software, Inc., San Diego, CA, USA).

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讨论

Extracellular matrix is an important ingredient for cell survival microenvironment, which depending on the interaction with the adaptor of cytomembrane to improve cell growth and cell attachment. Poly-L-ornithine is usually used as a component of extracellular matrix to simulate cell microenvironment during the cell culture in vitro^[23] . Studies about the effect of the extracellular matrix on biological activity of stem cells are still few. In this study we mainly evaluated the influence of Corning^® CellBIND^® Surface culture dish coated with poly-L-ornithine on cell cycle, cell proliferation, cell attachment and other characteristics. At present, there are few studies on observing the proliferation and adhesion of stem cells that are cultured in cell culture dish coated with poly-L-ornithine.

A suitable copolymer surface has been developed for cell culture^[24-30] . By comparing Corning^® CellBIND^®Surface culture dish with that coated with poly-L-ornithine, our results indicated that Corning^® CellBIND^®Surface culture dish promoted cell attachment and cell proliferation faster than Corning^®culture dishes. Importantly, cells cultured on the special surface show neither induced differentiation under standard growth conditions nor a rate of spontaneous differentiation greater than that seen with Corning^®tissue culture polystyrene dish as indicated by hUMSCs phenotypes of CD73, CD90 and CD105. And there are no advanced effects on cell proliferative protein expressions as indicated as cyclinA, cyclinE and PCNA. It means that by using microwave plasma process for treating the culture surface, Corning^®CellBIND^® Surface culture dish exerts the excellent effect on cell attachment and cell proliferation and maintains the characters of hUMSCs. Poly-L-ornithine coating does not change the character of Corning^® CellBIND^®Surface culture dish, and also develops the ability of Corning^® CellBIND^® Surface culture dish^[31-39] . It is for the first time suggested that the promotion effect of the extracellular matrix component- poly-L-ornithine on cell proliferation and cell adsorption. The effect excellently shown on Corning^® CellBIND^® Surface does not affect cell proliferation and cell adsorption, and promotes characteristics of Corning^®CellBIND^® Surface culture dish itself. However, the changes of hUMSCs behavior and differentiation after long-term culture in poly-L-ornithine coated culture dish need to be further studied.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
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