人皮膚成纖維細胞攪拌培養(yǎng)和保存的初步研究
發(fā)布時間：2018-11-20 15:34
【摘要】：目的:利用磁力攪拌培養(yǎng)器微載體攪拌培養(yǎng)人皮膚成纖維細胞,觀察細胞的生長增殖情況;檢測細胞增殖達到平臺期后不同保存條件下細胞的活力及保存時間;觀察攪拌培養(yǎng)微載體上細胞接種到普通培養(yǎng)瓶中移行、增殖和分泌Ⅰ型膠原含量的情況;觀察攪拌培養(yǎng)的人皮膚成纖維細胞用于制備復方殼多糖人工皮膚后的增殖情況及細胞形態(tài)。 方法:利用酶消化法分別培養(yǎng)出人包皮成纖維細胞和包皮表皮細胞,選取6～10代的人皮膚成纖維細胞(細胞量為8×10~6,密度為4×10~4/ml)接種到濃度為2.5g/L的微載體cytodex-3攪拌培養(yǎng)瓶中,用200ml改良培養(yǎng)基按20rpm/min,連續(xù)攪拌50min,暫停5min,循環(huán)模式進行攪拌培養(yǎng),以靜態(tài)培養(yǎng)為對照,利用CASY細胞計數(shù)儀檢測兩種方法培養(yǎng)每隔24h后細胞各自的增殖情況,倒置相差顯微鏡定時觀察和掃描電鏡選時相點觀察細胞形態(tài)并照相;檢測細胞培養(yǎng)增殖達到平臺期后的保存時間和MTT法檢測在室溫和4℃保存條件下細胞混懸液的OD值,據(jù)此計算出活細胞量制作活細胞量曲線;在攪拌培養(yǎng)第9天取微載體成纖維細胞混懸液15ml進行移行培養(yǎng)觀察,并做細胞爬片的vimentin免疫組化染色鑒定人皮膚成纖維細胞;用ELISA方法測定移入方瓶培養(yǎng)后培養(yǎng)液中Ⅰ型膠原的含量;按我科方法制備復方殼多糖組織工程皮膚,對比觀察攪拌培養(yǎng)和普通培養(yǎng)的成纖維細胞植入復方殼多糖組織工程皮膚后的增殖情況,進行HE染色觀察。 結果:人皮膚成纖維細胞接種在微載體上攪拌培養(yǎng)具有正常的形態(tài),與靜態(tài)培養(yǎng)比較,需要的微載體量少(2.5g/L),以此微載體濃度進行細胞擴增培養(yǎng),細胞總產(chǎn)量與接種量之間呈數(shù)量級擴增,大多數(shù)微載體上能長滿成纖維細胞,且需要的培養(yǎng)基量少(每瓶200ml改良培養(yǎng)基),能快速大規(guī)模擴增,從接種增殖到達平臺期只需10～12天,且收獲細胞量是接種量的5倍左右;在攪拌培養(yǎng)的人皮膚成纖維細胞達到增殖平臺期后,室溫和4℃條件下在微載體上保存的細胞活力比靜態(tài)培養(yǎng)的人皮膚成纖維細胞保存的時間長,室溫條件下能保存三周左右,在第3天時保存的細胞活力有較大一過性降低,隨后又逐漸恢復,活力在第19天時最佳,可達到86.96%,到第23天時細胞活力明顯下降只有64.36%,而4℃條件下相應保存的細胞活力到第19天時只有13.04%,細胞活力下降明顯,均低于室溫保存的細胞;攪拌培養(yǎng)后第9天移入方瓶中培養(yǎng)的細胞具有正常形態(tài)和增殖特性,收集不同時間點的細胞培養(yǎng)液測定Ⅰ型膠原含量結果顯示,在一定時間范圍內,Ⅰ型膠原含量前5天隨時間的推移和細胞的增殖而分泌增加;觀察混合成纖維細胞培養(yǎng)的復方殼多糖組織工程皮膚,結果顯示,2周左右HE染色可見人皮膚成纖維細胞增殖布滿真皮。 結論:選取微載體濃度為2.5g/L攪拌培養(yǎng)HDF是一種較為簡易、經(jīng)濟且迅速有效擴增細胞的方法;HDF經(jīng)攪拌培養(yǎng)后保持了良好的生物學特性;攪拌培養(yǎng)后HDF保存的時間比靜態(tài)的時間長,室溫下能保存3周左右;該保存方法簡單易行、經(jīng)濟實用,為種子細胞的運輸和保存開辟了一條新的思路;微載體上培養(yǎng)的HDF制備復合殼多糖組織工程皮膚形態(tài)良好,細胞增殖快速均勻。
[Abstract]:Objective: To observe the growth and proliferation of human skin fibroblasts by using a magnetic stirrer microcarrier to stir and culture human skin fibroblasts. The proliferation and the cell morphology of the cultured human skin fibroblasts were observed in the preparation of the compound shell-polysaccharide artificial skin. Methods: Human foreskin fibroblasts and prepuce epidermal cells were cultured by enzyme digestion, and 6-10 generations of human skin fibroblasts (8-10-6 cells, 4-10-4/ ml, 4-10-4/ ml, 4-10-4/ ml) were used to inoculate the culture flask with a microcarrier with a concentration of 2.5g/ L. In the process of continuous stirring for 50min, suspension for 5min, and the circulation mode was stirred and cultured in a static culture, the cell morphology of the cells was observed by the phase-point observation at the time of the phase-difference microscope and the scanning electron microscope by using the CASY cell counter to test the proliferation of the cells after every 24 h. photographing; detecting the storage time after the cell culture multiplication reaches the plateau period and the OD value of the cell suspension liquid at the room temperature and the storage condition at the temperature of 4 DEG C by the MTT method, curve; carrying out the migration culture observation on the microcarrier fibroblast suspension liquid 15ml on the 9th day of the stirring culture, and carrying out the vimentin immunohistochemical staining of the cell creeper to identify the human skin fibroblast; and measuring the type I collagen in the culture solution after the moving-in bottle culture by the ELISA method. Content: Prepare the compound shell polysaccharide tissue engineering skin according to the method of the family, compare and observe the proliferation of the mixed shell polysaccharide tissue engineering skin after the mixed culture and the normal culture, and carry out HE staining Results: The human skin fibroblasts were inoculated on the microcarrier, and the culture was in a normal form. Compared with the static culture, the amount of the microcarriers required was less (2.5g/ L), and the microcarrier concentration was used to carry out the cell amplification culture, and the total cell yield and the amount of inoculation were in the form of the microcarrier. on the order of magnitude, most of the microcarriers can be overgrown with fibroblasts, and the amount of culture medium required is small (per bottle of 200ml modified culture medium), and can be rapidly and massively expanded, only 10-12 days are required from the inoculation and propagation to the platform stage, and the amount of the harvested cells is the inoculation amount. and the cell activity stored on the microcarrier under the condition of room temperature and 4 DEG C is longer than that of the static cultured human skin fibroblasts after the cultured human skin fibroblasts reach the proliferation platform period, and can be protected under the condition of room temperature. The viability of the cells, which was preserved at day 3, was reduced by about three weeks, and then recovered gradually. The viability was the best at day 19 and reached 86.96%. By day 23, the cell viability was significantly reduced by only 64. 36%, while the corresponding cell viability at 4.degree. C. was only 1. 3.04%, the cell viability was significantly lower than that of the cells preserved at room temperature; the cells cultured on the 9th day after the stirring culture had the normal form and the proliferation characteristic, and the cell culture liquid collected at different time points was collected to determine the type I collagen content and the results showed that the cell culture medium In the time range, the concentration of type I collagen increased with the time and the proliferation of the cells. The skin of the compound shell polysaccharide was observed in the cell culture of the mixed synthetic fiber. The results showed that the human skin fibroblasts increased by 2 weeks of HE staining. Conclusion: The culture of HDF with the concentration of 2.5g/ L is a simple, economical and effective method for the rapid and effective amplification of the cells. The HDF has good biological characteristics after being stirred and cultured. The time of the HDF preservation after stirring and culture is longer than the static time, and the room temperature The preservation method is simple and feasible, is economical and practical, opens a new way for the transportation and preservation of the seed cells,
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329
本文編號：2345285
[Abstract]:Objective: To observe the growth and proliferation of human skin fibroblasts by using a magnetic stirrer microcarrier to stir and culture human skin fibroblasts. The proliferation and the cell morphology of the cultured human skin fibroblasts were observed in the preparation of the compound shell-polysaccharide artificial skin. Methods: Human foreskin fibroblasts and prepuce epidermal cells were cultured by enzyme digestion, and 6-10 generations of human skin fibroblasts (8-10-6 cells, 4-10-4/ ml, 4-10-4/ ml, 4-10-4/ ml) were used to inoculate the culture flask with a microcarrier with a concentration of 2.5g/ L. In the process of continuous stirring for 50min, suspension for 5min, and the circulation mode was stirred and cultured in a static culture, the cell morphology of the cells was observed by the phase-point observation at the time of the phase-difference microscope and the scanning electron microscope by using the CASY cell counter to test the proliferation of the cells after every 24 h. photographing; detecting the storage time after the cell culture multiplication reaches the plateau period and the OD value of the cell suspension liquid at the room temperature and the storage condition at the temperature of 4 DEG C by the MTT method, curve; carrying out the migration culture observation on the microcarrier fibroblast suspension liquid 15ml on the 9th day of the stirring culture, and carrying out the vimentin immunohistochemical staining of the cell creeper to identify the human skin fibroblast; and measuring the type I collagen in the culture solution after the moving-in bottle culture by the ELISA method. Content: Prepare the compound shell polysaccharide tissue engineering skin according to the method of the family, compare and observe the proliferation of the mixed shell polysaccharide tissue engineering skin after the mixed culture and the normal culture, and carry out HE staining Results: The human skin fibroblasts were inoculated on the microcarrier, and the culture was in a normal form. Compared with the static culture, the amount of the microcarriers required was less (2.5g/ L), and the microcarrier concentration was used to carry out the cell amplification culture, and the total cell yield and the amount of inoculation were in the form of the microcarrier. on the order of magnitude, most of the microcarriers can be overgrown with fibroblasts, and the amount of culture medium required is small (per bottle of 200ml modified culture medium), and can be rapidly and massively expanded, only 10-12 days are required from the inoculation and propagation to the platform stage, and the amount of the harvested cells is the inoculation amount. and the cell activity stored on the microcarrier under the condition of room temperature and 4 DEG C is longer than that of the static cultured human skin fibroblasts after the cultured human skin fibroblasts reach the proliferation platform period, and can be protected under the condition of room temperature. The viability of the cells, which was preserved at day 3, was reduced by about three weeks, and then recovered gradually. The viability was the best at day 19 and reached 86.96%. By day 23, the cell viability was significantly reduced by only 64. 36%, while the corresponding cell viability at 4.degree. C. was only 1. 3.04%, the cell viability was significantly lower than that of the cells preserved at room temperature; the cells cultured on the 9th day after the stirring culture had the normal form and the proliferation characteristic, and the cell culture liquid collected at different time points was collected to determine the type I collagen content and the results showed that the cell culture medium In the time range, the concentration of type I collagen increased with the time and the proliferation of the cells. The skin of the compound shell polysaccharide was observed in the cell culture of the mixed synthetic fiber. The results showed that the human skin fibroblasts increased by 2 weeks of HE staining. Conclusion: The culture of HDF with the concentration of 2.5g/ L is a simple, economical and effective method for the rapid and effective amplification of the cells. The HDF has good biological characteristics after being stirred and cultured. The time of the HDF preservation after stirring and culture is longer than the static time, and the room temperature The preservation method is simple and feasible, is economical and practical, opens a new way for the transportation and preservation of the seed cells,
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329
【參考文獻】
相關期刊論文 前10條
1 張鳳菊,劉超,成國祥;聚合物微載體及其在細胞培養(yǎng)方面的應用[J];北京生物醫(yī)學工程;2003年03期
2 關英杰,魏少敏,金錫鵬;皮膚細胞培養(yǎng)技術在化妝品功效研究中的應用[J];日用化學工業(yè);2002年05期
3 伍津津,朱堂友,魯元剛,楊宏珍;表皮真皮分離方法的探索[J];第三軍醫(yī)大學學報;2004年24期
4 張立,嚴春,范衛(wèi)民,張元興,俞俊棠;Vero細胞的微載體培養(yǎng)——放大過程中的接種工藝[J];華東理工大學學報;1998年06期
5 薛毅瓏;重視微囊化人工細胞在醫(yī)學中應用的研究[J];解放軍醫(yī)學雜志;1999年04期
6 李潔,劉斌,李焰,婁超,婁皓;不同條件下細胞凍存效果比較[J];臨床口腔醫(yī)學雜志;2004年02期
7 王曦,李利;成纖維細胞與皮膚老化[J];中國美容醫(yī)學;2005年02期
8 張誠;王一兵;張芮;李昊;馮永強;;大鼠胚胎皮膚成纖維細胞移植的研究[J];山東醫(yī)藥;2006年14期
9 王常勇;采用微載體技術大規(guī)模培養(yǎng)組織工程種子細胞[J];生物醫(yī)學工程與臨床;2002年01期
10 鄧繼先,楊琴,程萱,李琳,周江;用無血清培養(yǎng)基在填充床生物反應器生產(chǎn) rHuEPO[J];生物工程學報;1997年04期
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