研究生: |
陳杰陞 Chen,Jie Sheng |
---|---|
論文名稱: |
腎足細胞培養於鍶離子固化3D水膠管狀結構 Strontium Ion Mediated Alginate Tubes for Highly-Viable 3D Core-Shell Glomerular Podocyte Cells Cultivation |
指導教授: |
曾繁根
Tseng,Fan Gang 王翔郁 Wang,Hsiang Yu |
口試委員: |
王本誠
Wang,Pen Cheng 許翔皓 Hsu,Hsiang Hao |
學位類別: |
碩士 Master |
系所名稱: |
原子科學院 - 工程與系統科學系 Department of Engineering and System Science |
論文出版年: | 2016 |
畢業學年度: | 104 |
語文別: | 中文 |
論文頁數: | 71 |
中文關鍵詞: | 腎足細胞 、海藻酸鈉 、鍶離子 、腎小球 |
外文關鍵詞: | podocyte, alginate, strontium, glomerular |
相關次數: | 點閱:2 下載:0 |
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在人體血液過濾當中,腎小球扮演著十分重要的角色。腎小球由內而外分別是內皮細胞、腎小球基底膜和腎足細胞。三者分別扮演著不同孔洞大小的過濾膜來過濾血液中需要留下來的東西,像是內皮細胞不讓紅血球通過、腎足細胞限制了血紅素或是白蛋白的流出。因此我們必須做出管狀且多層共軸結構,成功的讓管子由內而外培養內皮細胞、腎小球基底膜和腎足細胞,才能模仿人體內腎小球的行為原理。過去的研究當中,要做成長條形的管狀共軸結構,外層都是用水膠,利用水膠碰到氯化鈣水溶液,水溶液當中的鈣離子會置換海藻酸鈉中的鈉離子產生交聯作用而固化來維持管狀的機械強度,內層通常都是以明膠混合著所要培養的細胞,內層外層同時用注射式幫浦注射而做出共軸雙層的結構。在第一階段的研究當中,我們致力於壓力對於腎足細胞的平面影響,畢竟台灣洗腎人口居高不下,洗腎密度高居世界前五,而高血壓又是洗腎的主要原因之一,因此先行在平面探討壓力對腎足細胞過濾效果的病理影響。有了第一階段的病理結果,在第二階段,我們重點放在尋找三維管狀結構的各種參數,包含製造三維管子的各種流速、材料種類、材料濃度等等。畢竟細胞在三維立體結構中,細胞的生長情況本來就不如在平面結構,而且在管中,細胞的生長環境不只有培養液,還有水膠以及交聯用的二價金屬離子,這些對細胞的生長都存在著影響。因此本階段就是在二維環境中尋找最佳參數以運用到三維管子的製造。至於最後一個階段則是實際製造出三維的管子。在實驗中發現腎足細胞不喜歡有氯化鈣的環境,因此我們進一步尋找其他二價離子使水膠固化。發現鍶離子不但對腎足細胞無害,能使腎足細胞進行分裂的行為,這一個步驟是腎小球內過濾的關鍵因素,除此之外,氯化鍶能讓水膠有更好的機械強度,這也為實驗未來接管的部分找到更佳的材料。
目前先以單一細胞去做實驗,未來的目標是做出三層結構以模仿腎小球內部的構造,而且內層內皮細胞以及外層腎足細胞所需要的生長環境也不太相同,因此需要在兩者之間取一平衡點讓兩者都能順利生長的最佳環境。
Glomerular play an important role of blood filtration in human body。From outside to inside are endothelia cells, glomerular basement membrane(GBM) and podocyte, respectively. These three kinds of cells act as the different size of filtration membranes to filter. For example, endothelia cells stop red blood cells crossing the blood vessel. Therefore we have to manufacture a core-shell multilayer co-axis microfibers, and culture three kinds of cell on the each layer. So that we can imitate the behavior of glomerular in human boddy.
In the past research, alginate is the only material of the co-axis microfibers. Alginate will have a cross-linking reaction when we add calcium chloride solution in it. The sodium ion in alginate will be replaced by the calcium ion. The hard alginate have to hold the microfibers by its mechanical strength. The material of the inside tube is gelatin with suspended cells. We use two syringe pump to produce the outside tube and inside tube at the same time. In the first step of our experiment, we focus on the influence of podocyte after giving pressure differences. The dialysis population still high is Taiwan. The density of dialysis population ranks in top five in all over the world. And hypertension is one of the leading causes of dialysis. So we do some research on podocyte filtration after giving pressure. With the pathological behavior and results in first step, we find out some best parameters including gel inject velocity, gel species, gel concentration in second step. Cells is not easily to survive in 3D structures. In 3D structures, there are many impact factors. They will influence the survival ratio of cells. In this step, we focus on finding the best environment for cells to survive. And supply these parameters to 3D structure cells culturing. In the last step, we manufacture 3D cell tube. In this period, we observe that podocyte are sensitive to calcium ion. So we cure alginate with strontium, instead. Podocyte stretch out under the environment of strontium. It is the key factor of glomerular filtration. Besides strontium cured alginate has a more stronger mechanical strength. This is the right material for the 3D cell tube.
For the future work, multi-layer 3D cell tube with endothelial cell, GBM and podocyte is our final purpose to mimic the glomerulr in vitro.
[1] Griffith, Linda G., and Gail Naughton. "Tissue engineering--current challenges and expanding opportunities." Science 295.5557 (2002): 1009-1014.
[2] Shin, Heungsoo, Seongbong Jo, and Antonios G. Mikos. "Biomimetic materials for tissue engineering." Biomaterials 24.24 (2003): 4353-4364.
[3]Yang, Shoufeng, et al. "The design of scaffolds for use in tissue engineering. Part I. Traditional factors." Tissue engineering 7.6 (2001): 679-689.
[4]Brunski, J.B. Metals. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., et al., eds. Biomaterials Science: An Introduction to Materials in Medicine. New York: Academic Press,(1996):37–50.
[5]Hench, L.L. Ceramics, glasses, and glass-ceramics. In: Ratner, B.D., Hoffman, A.S., Schoen, F.J., et al., eds. Biomaterials Science: An Introduction to Materials in Medicine. New York: Academic Press, (1996):73–83.
[6]Mano, J. F., et al. "Dynamic mechanical properties of hydroxyapatite-reinforced and porous starch-based degradable biomaterials." Journal of Materials Science: Materials in Medicine 10.12 (1999): 857-862.
[7] Carlsson, Roland, et al. "Laminin and fibronectin in cell adhesion: enhanced adhesion of cells from regenerating liver to laminin." Proceedings of the National Academy of Sciences 78.4 (1981): 2403-2406.
[8] Rich, A. K. H. A., and Albert K. Harris. "Anomalous preferences of cultured macrophages for hydrophobic and roughened substrata." Journal of cell science50.1 (1981): 1-7.
[9] Sheetz, Michael P., Dan P. Felsenfeld, and Catherine G. Galbraith. "Cell migration: regulation of force on extracellular-matrix-integrin complexes."Trends in cell biology 8.2 (1998): 51-54.
[10] Meyle, J., et al. "Fibroblast anchorage to microtextured surfaces." Journal of biomedical materials research 27.12 (1993): 1553-1557.
[11] Meyle, J., et al. "Fibroblast anchorage to microtextured surfaces." Journal of biomedical materials research 27.12 (1993): 1553-1557.
[12Singhvi, Rahul, et al. "Engineering cell shape and function." SCIENCE-NEW YORK THEN WASHINGTON- (1994): 696-696. [13] Chou, Laisheng, et al. "Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts." Journal of Cell Science 108.4 (1995): 1563-1573.
[14] Peppas, Nicholas A., ed. Hydrogels in medicine and pharmacy: properties and applications. Vol. 3. CRC PressI Llc, 1987. [15]Kvantinių taškų panaudojimas nanomedicinoje. Dr. V. Karabanovas. Kaunas, (2012).
[16]Scranton AB, Bowman CN, et al. photopolymerization fundamentals and applications. New Orleans: ACS Publishers, (1996).
[17] Qiu, Yong, and Kinam Park. "Environment-sensitive hydrogels for drug delivery." Advanced drug delivery reviews 53.3 (2001): 321-339.
[18] Bushetti, S. S., et al. "Stimuli sensitive hydrogels: a review." Indian Journal of Pharmaceutical Education and Research 43.3 (2009): 241-250.
[19] Slaughter, Brandon V., et al. "Hydrogels in regenerative medicine." Advanced materials 21.32‐33 (2009): 3307-3329. [20] Xu, Yuanting, et al. "Feasibility study of a novel crosslinking reagent (alginate dialdehyde) for biological tissue fixation." Carbohydrate polymers 87.2 (2012): 1589-1595.
[21] Topuz, Fuat, et al. "Magnesium ions and alginate do form hydrogels: a rheological study." Soft Matter 8.18 (2012): 4877-4881. [22] Place, Elsie S., et al. "Strontium-and zinc-alginate hydrogels for bone tissue engineering." Tissue Engineering Part A 17.21-22 (2011): 2713-2722.
[23] Yamaguchi, Yu, David M. Mann, and Erkki Ruoslahti. "Negative regulation of transforming growth factor-beta by the proteoglycan decorin." Nature 346.6281 (1990): 281-284.
[24] Schuppan, Detlef, et al. "Collagens in the liver extracellular matrix bind hepatocyte growth factor." Gastroenterology 114.1 (1998): 139-152.
[25] Hulmes, D. J., and A. N. D. R. E. W. Miller. "Molecular packing in collagen."Nature 293 (1981): 239-234.
[26] Shapiro, Jenna M., and Michelle L. Oyen. "Hydrogel composite materials for tissue engineering scaffolds." Jom 65.4 (2013): 505-516.
[27] Ignatius, Anita, et al. "Tissue engineering of bone: effects of mechanical strain on osteoblastic cells in type I collagen matrices." Biomaterials 26.3 (2005): 311-318.
[29] Adekogbe, Iyabo, and Amyl Ghanem. "Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering." Biomaterials26.35 (2005): 7241-7250.
[30] Huang, Yan, et al. "In vitro characterization of chitosan–gelatin scaffolds for tissue engineering." Biomaterials 26.36 (2005): 7616-7627.
[31] Li, Zhensheng, et al. "Chitosan–alginate hybrid scaffolds for bone tissue engineering." Biomaterials 26.18 (2005): 3919-3928. [32] Li, Xudong, et al. "Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering." Biomaterials 27.11 (2006): 2426-2433.
[33] Mi, Fwu-Long, et al. "In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant." Biomaterials 23.1 (2002): 181-191.
[34] Cremers, H. F. M., et al. "Albumin-heparin microspheres as carriers for cytostatic agents." Journal of controlled release 11.1 (1990): 167-179.
[35] Tabata, Yasuhiko, et al. "Skull bone regeneration in primates in response to basic fibroblast growth factor." Journal of neurosurgery 91.5 (1999): 851-856.
[36] Ozekp, Makoto, et al. "Controlled release of hepatocyte growth factor from gelatin hydrogels based on hydrogel degradation." Journal of drug targeting 9.6 (2001): 461-471.
[37] Hoffman, Allan S. "Hydrogels for biomedical applications." Advanced drug delivery reviews 64 (2012): 18-23.
[38] Drury, Jeanie L., and David J. Mooney. "Hydrogels for tissue engineering: scaffold design variables and applications." Biomaterials 24.24 (2003): 4337-4351.
[39] Tibbitt, Mark W., and Kristi S. Anseth. "Hydrogels as extracellular matrix mimics for 3D cell culture." Biotechnology and bioengineering 103.4 (2009): 655-663.
[40] Hsieh, Hsin-Yi, et al. "Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment." Lab on a Chip 14.3 (2014): 482-493.
[41] Naito, Hiroshi, et al. "The advantages of three‐dimensional culture in a collagen hydrogel for stem cell differentiation." Journal of Biomedical Materials Research Part A 101.10 (2013): 2838-2845.
[42] Jongpaiboonkit, Leenaporn, William J. King, and William L. Murphy. "Screening for 3D environments that support human mesenchymal stem cell viability using hydrogel arrays." Tissue Engineering Part A 15.2 (2008): 343-353.
[43] FW Greiner, Johannes, et al. "Going 3D–Cell Culture Approaches for Stem Cell Research and Therapy." Current Tissue Engineering 2.1 (2013): 8-19.
[44] Barralet, J. E., et al. "Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels." Journal of materials science: materials in medicine 16.6 (2005): 515-519.
[45] Rowley, Jon A., Gerard Madlambayan, and David J. Mooney. "Alginate hydrogels as synthetic extracellular matrix materials." Biomaterials 20.1 (1999): 45-53.
[46] Pavenstädt, Hermann, Wilhelm Kriz, and Matthias Kretzler. "Cell biology of the glomerular podocyte." Physiological reviews 83.1 (2003): 253-307.
[47]呂芷萱. "具奈米孔洞仿生膜之微流體系統應用於研究壓力對腎臟足細胞生理以及病理上的影響." 清華大學奈米工程與微系統研究所學位論文 (2014): 1-69
[48] Tottori, Soichiro, and Shoji Takeuchi. "Formation of liquid rope coils in a coaxial microfluidic device." RSC Advances 5.42 (2015): 33691-33695.
[49] Onoe, Hiroaki, et al. "Metre-long cell-laden microfibres exhibit tissue morphologies and functions." Nature materials 12.6 (2013): 584-590.
[50] Yoon, D. H., et al. "One core-five sheaths coaxial flow formation using multilayer stacked flow focusing structure." 2015 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS). IEEE, (2015).