旋轉(zhuǎn)式生物反應(yīng)器內(nèi)微載體培養(yǎng)擴增皮膚相關(guān)細胞及微粒皮膚的初步制備
發(fā)布時間：2019-05-16 22:57
【摘要】：隨著細胞培養(yǎng)技術(shù)的發(fā)展,對皮膚相關(guān)細胞的培養(yǎng),特別是人表皮細胞的培養(yǎng)有了很大的進展。在適宜的培養(yǎng)條件下,單個細胞在培養(yǎng)中不斷增殖,逐漸融合成片狀,形成了類似皮膚表皮層的上皮組織。這使得在較短的時間里,用大量表皮細胞培育成小片狀的上皮組織成為可能。臨床工作中,外科醫(yī)生和細胞生物學(xué)家們也大量培養(yǎng)自體表皮細胞并用來修復(fù)皮膚缺損的病人。表皮細胞培養(yǎng)技術(shù)也從使用含血清培養(yǎng)液和細胞滋養(yǎng)層的培養(yǎng)方式,到不含血清、無滋養(yǎng)層的培養(yǎng)方式。培養(yǎng)方法的發(fā)展也促進了臨床應(yīng)用,從最初的細胞懸液移植到自體異體細胞膜片移植,以及表皮細胞生物材料復(fù)合物移植。培養(yǎng)和移植方法的發(fā)展使人們在治療大面積皮膚缺損方面看到了希望。 細胞培養(yǎng)技術(shù)對研究動物細胞的結(jié)構(gòu)、功能和分化過程中的作用非常重要。因此,將微載體培養(yǎng)技術(shù)應(yīng)用到貼壁依賴型細胞的大量培養(yǎng),使上述研究進入到了一個新的層次。在微載體培養(yǎng)環(huán)境中,通過在培養(yǎng)液中輕柔的轉(zhuǎn)動或者攪拌,細胞在微球表面呈現(xiàn)單層或者復(fù)層生長,在這種無復(fù)雜結(jié)構(gòu)的懸浮培養(yǎng)體系中,可獲得2x10~8個/毫升的細胞量。 Cytodex微載體是GE Healthcare公司為大量培養(yǎng)各類動物細胞專門開發(fā)的微載體,其培養(yǎng)體系的容器可以從數(shù)毫升到數(shù)千升,非常適合大規(guī)模擴增細胞。這種微載體的表面對細胞生長過程中的貼附和伸展過程專門優(yōu)化,它的直徑和密度也非常適合懸浮狀態(tài)大規(guī)模培養(yǎng)擴增各類細胞。構(gòu)成微載體球體的基質(zhì)材料生物穩(wěn)定性很好,提供給細胞適宜的表面生長環(huán)境。透光的球體也很容易通過顯微鏡觀察貼附之上的細胞。通過以往的廣泛使用可以看出,cytodex十分適合作為微載體培養(yǎng)細胞。 旋轉(zhuǎn)式生物反應(yīng)器是由一個水平軸旋轉(zhuǎn)的培養(yǎng)容器和與其共軸的氧合器組成,當(dāng)容器以水平軸開始旋轉(zhuǎn)時,充滿其中的培養(yǎng)液作為一個整體也發(fā)生旋轉(zhuǎn),這種方式使內(nèi)容物受到極小的剪切力。培養(yǎng)體系中的細胞旋轉(zhuǎn)時所受的離心力、重力和科氏力抵消,因此細胞能大體維持懸浮狀態(tài)。培養(yǎng)體系中的細胞受到極小的機械力的同時獲得大量的氧、營養(yǎng)物質(zhì)的供應(yīng)。細胞所需的氣體通過硅橡膠膜微量而持續(xù)的交換,避免了旋轉(zhuǎn)培養(yǎng)體系中出現(xiàn)大的氣泡和渦流。 在臨床工作中,使用自體表皮,成纖維細胞快速有效的修復(fù)各類皮膚缺損對皮膚愈合有很大意義,而足夠數(shù)量的種子細胞對構(gòu)建組織工程皮膚非常重要。本研究嘗試使用Cytodex-3微載體和高截面縱橫比的旋轉(zhuǎn)式生物反應(yīng)器作為培養(yǎng)系統(tǒng),大規(guī)模擴增人真皮成纖維細胞和人表皮細胞,期待能為各類組織工程皮膚產(chǎn)品提供充足的種子細胞。 第一部分實驗成纖維細胞和表皮細胞的體外培養(yǎng)及鑒定 1主要方法 獲取新生兒包皮切除術(shù)后組織,人表皮細胞使用中性蛋白酶和胰蛋白酶兩步消化法獲取;人真皮成纖維細胞使用胰酶消化組織塊貼壁法獲取。待原代培養(yǎng)成功后,常規(guī)傳代。免疫組化方法進行抗波形絲蛋白、抗角蛋白染色,顯微鏡下觀察。 2主要結(jié)果 倒置顯微鏡下觀察細胞形態(tài)和生長情況。人表皮細胞呈圓形,細胞鋪滿培養(yǎng)瓶底,密度均勻。人真皮成纖維細胞呈長梭形,胞核飽滿。免疫組化結(jié)果顯示人真皮成纖維細胞抗波形絲蛋白染色陽性,人表皮細胞抗角蛋白染色為陽性。 3主要結(jié)論 通過鑒定,本實驗所獲取以及體外培養(yǎng)擴增的細胞分別為人真皮成纖維細胞和人表皮細胞。 第二部分實驗旋轉(zhuǎn)式生物反應(yīng)器培養(yǎng)擴增人真皮成纖維細胞 1主要方法 利用組織貼壁法從人皮膚中分離出人真皮成纖維細胞(hDFBs)進行體外培養(yǎng),使用DIO標(biāo)記細胞后結(jié)合微載體在旋轉(zhuǎn)式生物反應(yīng)器(RCCS)中培養(yǎng),細胞貼附微載體的狀況使用熒光顯微鏡,掃描電鏡觀測。并且檢測細胞周期和分析細胞群體倍增時間來比較微重力培養(yǎng)與平面培養(yǎng)的體外增殖能力差異。 2主要結(jié)果 在旋轉(zhuǎn)式生物反應(yīng)器的微重力培養(yǎng)體系中,人成纖維細胞能快速貼附到微載體表面,在培養(yǎng)過程中達到很大的細胞密度,并且表現(xiàn)出很強的增殖能力和細胞活性。 3主要結(jié)論 利用生物反應(yīng)器和微載體懸浮培養(yǎng)人皮膚成纖維細胞,是大量制備皮膚組織工程種子細胞的一種有效方法。 第三部分實驗使用旋轉(zhuǎn)式生物反應(yīng)器體外擴增人表皮細胞 1主要方法 使用Cytodex-3微載體和高截面縱橫比的旋轉(zhuǎn)式生物反應(yīng)器容器作為培養(yǎng)系統(tǒng)大規(guī)模擴增人表皮細胞(hECs)。用中性蛋白酶和胰蛋白酶-EDTA兩步驟法從人皮膚中分離出人表皮細胞,使用DIL標(biāo)記細胞后結(jié)合微載體后在旋轉(zhuǎn)式生物反應(yīng)器(RCCS)中培養(yǎng),細胞貼附微載體的生長狀態(tài)使用倒置顯微鏡,掃描電鏡觀測。并且分析細胞群體倍增時間來比較微重力培養(yǎng)與平面培養(yǎng)的體外增殖能力差異。 2主要結(jié)果 在旋轉(zhuǎn)式生物反應(yīng)器的微重力培養(yǎng)體系中,人表皮細胞能快速貼附到微載體表面,在培養(yǎng)過程中達到很大的細胞密度,并且表現(xiàn)出很強的增殖能力和細胞活性。 3主要結(jié)論 使用旋轉(zhuǎn)式生物反應(yīng)器和微載體懸浮培養(yǎng)人表皮細胞,是大量制備皮膚組織工程種子細胞的一種有效方法。 第四部分實驗使用旋轉(zhuǎn)式生物反應(yīng)器制備微粒皮膚 1主要方法 人表皮細胞的體外培養(yǎng)中,培養(yǎng)液中的血清成分有誘導(dǎo)分化作用,會使其過早的進入終末狀態(tài),增殖趨于停止,通常對表皮細胞的培養(yǎng)早期使用角質(zhì)細胞無血清培養(yǎng)液(K-SFM )。而在人成纖維細胞在體外培養(yǎng)過程中,含血清的培養(yǎng)液對其增殖有利。考慮到兩種細胞對培養(yǎng)液的不同要求,本實驗分別嘗試兩種不同方法:培養(yǎng)液血清濃度梯度降低法和表皮培養(yǎng)液共培養(yǎng)法,使用Cytodex-3微載體和高截面縱橫比的旋轉(zhuǎn)式生物反應(yīng)器容器作為培養(yǎng)系統(tǒng)嘗試制備微粒皮膚。并使用掃描電鏡觀察細胞的貼附生長狀態(tài)。 2主要結(jié)果 表皮培養(yǎng)液共培養(yǎng)法中,微載體顆粒上有多角形的表皮細胞貼附,成纖維細胞貼附狀態(tài)較差,部分從微載體上脫落。血清濃度梯度降低法中,微載體上有多角形的表皮細胞和梭形的成纖維細胞貼附,細胞貼附狀態(tài)較好,但細胞密度較小。 3主要結(jié)論 使用旋轉(zhuǎn)式生物反應(yīng)器和微載體懸浮培養(yǎng)微粒皮膚,培養(yǎng)液的血清濃度對共培養(yǎng)的兩種細胞影響較大,使用血清濃度梯度降低法的共培養(yǎng)體系效果較好。
[Abstract]:With the development of cell culture technology, the culture of skin-related cells, especially the culture of human epidermal cells, has made great progress. Under suitable culture conditions, a single cell is continuously proliferating in culture and is gradually fused into a sheet form, and an epithelial tissue similar to the skin layer of the skin is formed. This makes it possible to develop a small sheet of epithelial tissue with a large amount of epidermal cells in a shorter period of time. In clinical work, surgeons and cell biologists also have a large number of patients who have cultured autologous epidermal cells and used to repair skin defects. The epidermal cell culture technique is also derived from the culture of the use of a serum-containing culture solution and a cell trophoblast, to a culture that does not contain serum and trophoblast. The development of the culture method also facilitates the clinical application, from the initial cell suspension to the autograft cell membrane graft, and the epidermal cell biological material complex transplantation. The development of the method of culture and transplantation has made people see the hope in the treatment of large-area skin defects. The cell culture technique plays a very important role in the study of the structure, function and differentiation of animal cells in order to do so, the microcarrier culture technique is applied to a large number of culture of the adherent-dependent cells, allowing the above-mentioned studies to come into a new layer Times. in a microcarrier culture environment, the cells present a monolayer or multi-layer growth on the surface of the microspheres by gentle rotation or agitation in the culture medium, in which 2x10 to 8/ ml of cells can be obtained in a suspension culture system without a complex structure. The Cytodex microcarrier is a microcarrier specially developed by GE Healthcare for a large number of animal cells. The container of the culture system can be from several milliliters to several thousand liters, and is very suitable for large-scale expansion. The surface of the microcarrier is specially optimized for the application and extension of the cell growth process, and the diameter and the density of the microcarrier are also very suitable for large-scale culture and amplification of the suspension state. The matrix material that forms the microsphere of the microcarrier is good in biological stability and is provided to the appropriate surface of the cell. A long environment. Light-transmitting spheres are also easily observed by a microscope. The cell, which is widely used in the past, can be seen to be very suitable as the micro-carrier culture. The rotary bioreactor is composed of a culture vessel which is rotated by a horizontal axis and an oxygenator coaxial with it, Small shear forces. The centrifugal force, gravity, and Coriolis force against the rotation of the cells in the culture system are set off, so that the cells can be generally in a suspension state. The cells in the culture system are subjected to a very small mechanical force while obtaining a large amount of oxygen, nutrition, The supply of the substance. The gas required for the cell is continuously exchanged through the micro-and continuous exchange of the silicone rubber membrane, thus avoiding the large occurrence of the rotating culture system. The rapid and effective repair of various skin defects in clinical work is of great significance to the healing of the skin, and a sufficient number of seed cells are used for the construction of the tissue engineer. It is very important to use Cytodex-3 microcarrier and high-section aspect ratio rotary bioreactor as a culture system for large-scale amplification of human dermal fibroblasts and human epidermal cells, and it is expected to provide various tissue engineering skin products Adequate seed cells. The first part of the experimental fibroblasts and the epidermis in vitro The primary method of culture and identification is to obtain the post-circumcision tissue of the newborn, and the human epidermal cell uses the neutral protein. obtaining by two-step digestion method of enzyme and trypsin; human dermal fibroblasts The tissue mass was digested with pancreatin. after the primary culture is successful, the conventional passaging is carried out; the anti-waveform silk protein is carried out by the immunohistochemistry method, anti-keratin dyeing to be observed under a microscope. The main results Observation of the morphology and growth of cells under an inverted microscope. Human epidermal cells Round, the cell is full of the culture bottle bottom, the density is even. Human dermal fibroblasts were long-shuttle, and the nucleus was full. The immunohistochemical results showed that the human dermal fibroblasts were resistant to the wave-like silk protein staining. Positive, person's watch The anti-keratin staining of the skin cells was positive. The main conclusions were as follows: the identification, the acquisition of the experiment and the in vitro culture and expansion. The increased cells are human dermal fibroblasts and human epidermal cells, respectively. partial experimental rotation The main method of culture and amplification of human dermal fibroblasts by rotary bioreactor is to use the method of tissue apposition Human dermal fibroblasts (hDFBs) isolated from the skin were cultured in vitro, and the cells were labeled with DIO and the microcarriers were combined in a rotary bioreactor (RCCS). The cell-attached microcarriers were cultured with a fluorescence microscope and a scanning electron microscope, and the cell cycle and the analysis of the cell population were detected. volume doubling time in the microgravity culture system of the rotary bioreactor, the human fibroblasts can be quickly attached to the surface of the microcarrier, and in the tissue culture system, the human fibroblasts can be quickly attached to the surface of the microcarrier, in the process of raising to a large cell density and exhibit a strong proliferative and cellular activity.3 The main conclusions are the use of a bioreactor and a microcarrier The suspension culture human skin fibroblast is a large number of engineering species for preparing skin tissue subcellular one in that third part, a primary method for the in vitro amplification of human epidermal cell 1 using a rotary bioreactor is use Cytodex-3 microcarrier The invention relates to a large-scale amplification human epidermal cell (hECs) of a culture system, which is a rotary type bioreactor container with high cross-section aspect ratio. The human epidermal cells are isolated from human skin by using a neutral protease and a trypsin-EDTA two-step method, culturing in a bioreactor (RCCS), and using an inverted microscope to attach the growth state of the cell to the microcarrier; the view of scanning electron microscope the cell population doubling time was analyzed to compare the difference of the in vitro proliferation of the microgravity culture and the plane culture. The main results were in the microgravity culture system of the rotary bioreactor, human epidermal cells can be quickly attached To the surface of the microcarrier, a large cell density is achieved during the culture, and a strong proliferative and cellular activity is shown. on the use of a rotary bioreactor and a microcarrier suspension culture epidermal cells, It is an effective method for preparing the skin tissue engineering seed cells in a large amount. The fourth part of the experiment uses a rotary bioreactor to prepare the particulate skin. 1. In vitro culture of human epidermal cells, the serum components in the culture solution are induced It can lead to a premature entry to a terminal state, and the proliferation tends to stop, usually for epidermal cells. In the early stage of culture, a serum-free culture medium (K-SFM) was used in the culture of human fibroblasts. interest. In view of the different requirements of the two cells for the culture solution, two different methods are tried in this experiment: the method of reducing the concentration gradient of the serum in the culture solution and the table The culture medium co-culture method, using Cytodex- 3 microcarriers and A rotary bioreactor vessel with high cross-section aspect ratio was used as a culture system to prepare the skin of the microparticles. The growth state of the cells was observed by scanning electron microscope. in that co-culture method of the main result, the micro-carrier particles have polygonal epidermal cells attached to the micro-carrier particle, the adhesion state of the fibroblasts is poor, microcarrier in that method of reduce the serum concentration gradient, the micro-carrier has a polygonal epidermal cell and a shuttle-shaped fibroblast, and the cell adhesion state is good, but the cell density is small.3 main conclusions are as follows:
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
本文編號：2478615
[Abstract]:With the development of cell culture technology, the culture of skin-related cells, especially the culture of human epidermal cells, has made great progress. Under suitable culture conditions, a single cell is continuously proliferating in culture and is gradually fused into a sheet form, and an epithelial tissue similar to the skin layer of the skin is formed. This makes it possible to develop a small sheet of epithelial tissue with a large amount of epidermal cells in a shorter period of time. In clinical work, surgeons and cell biologists also have a large number of patients who have cultured autologous epidermal cells and used to repair skin defects. The epidermal cell culture technique is also derived from the culture of the use of a serum-containing culture solution and a cell trophoblast, to a culture that does not contain serum and trophoblast. The development of the culture method also facilitates the clinical application, from the initial cell suspension to the autograft cell membrane graft, and the epidermal cell biological material complex transplantation. The development of the method of culture and transplantation has made people see the hope in the treatment of large-area skin defects. The cell culture technique plays a very important role in the study of the structure, function and differentiation of animal cells in order to do so, the microcarrier culture technique is applied to a large number of culture of the adherent-dependent cells, allowing the above-mentioned studies to come into a new layer Times. in a microcarrier culture environment, the cells present a monolayer or multi-layer growth on the surface of the microspheres by gentle rotation or agitation in the culture medium, in which 2x10 to 8/ ml of cells can be obtained in a suspension culture system without a complex structure. The Cytodex microcarrier is a microcarrier specially developed by GE Healthcare for a large number of animal cells. The container of the culture system can be from several milliliters to several thousand liters, and is very suitable for large-scale expansion. The surface of the microcarrier is specially optimized for the application and extension of the cell growth process, and the diameter and the density of the microcarrier are also very suitable for large-scale culture and amplification of the suspension state. The matrix material that forms the microsphere of the microcarrier is good in biological stability and is provided to the appropriate surface of the cell. A long environment. Light-transmitting spheres are also easily observed by a microscope. The cell, which is widely used in the past, can be seen to be very suitable as the micro-carrier culture. The rotary bioreactor is composed of a culture vessel which is rotated by a horizontal axis and an oxygenator coaxial with it, Small shear forces. The centrifugal force, gravity, and Coriolis force against the rotation of the cells in the culture system are set off, so that the cells can be generally in a suspension state. The cells in the culture system are subjected to a very small mechanical force while obtaining a large amount of oxygen, nutrition, The supply of the substance. The gas required for the cell is continuously exchanged through the micro-and continuous exchange of the silicone rubber membrane, thus avoiding the large occurrence of the rotating culture system. The rapid and effective repair of various skin defects in clinical work is of great significance to the healing of the skin, and a sufficient number of seed cells are used for the construction of the tissue engineer. It is very important to use Cytodex-3 microcarrier and high-section aspect ratio rotary bioreactor as a culture system for large-scale amplification of human dermal fibroblasts and human epidermal cells, and it is expected to provide various tissue engineering skin products Adequate seed cells. The first part of the experimental fibroblasts and the epidermis in vitro The primary method of culture and identification is to obtain the post-circumcision tissue of the newborn, and the human epidermal cell uses the neutral protein. obtaining by two-step digestion method of enzyme and trypsin; human dermal fibroblasts The tissue mass was digested with pancreatin. after the primary culture is successful, the conventional passaging is carried out; the anti-waveform silk protein is carried out by the immunohistochemistry method, anti-keratin dyeing to be observed under a microscope. The main results Observation of the morphology and growth of cells under an inverted microscope. Human epidermal cells Round, the cell is full of the culture bottle bottom, the density is even. Human dermal fibroblasts were long-shuttle, and the nucleus was full. The immunohistochemical results showed that the human dermal fibroblasts were resistant to the wave-like silk protein staining. Positive, person's watch The anti-keratin staining of the skin cells was positive. The main conclusions were as follows: the identification, the acquisition of the experiment and the in vitro culture and expansion. The increased cells are human dermal fibroblasts and human epidermal cells, respectively. partial experimental rotation The main method of culture and amplification of human dermal fibroblasts by rotary bioreactor is to use the method of tissue apposition Human dermal fibroblasts (hDFBs) isolated from the skin were cultured in vitro, and the cells were labeled with DIO and the microcarriers were combined in a rotary bioreactor (RCCS). The cell-attached microcarriers were cultured with a fluorescence microscope and a scanning electron microscope, and the cell cycle and the analysis of the cell population were detected. volume doubling time in the microgravity culture system of the rotary bioreactor, the human fibroblasts can be quickly attached to the surface of the microcarrier, and in the tissue culture system, the human fibroblasts can be quickly attached to the surface of the microcarrier, in the process of raising to a large cell density and exhibit a strong proliferative and cellular activity.3 The main conclusions are the use of a bioreactor and a microcarrier The suspension culture human skin fibroblast is a large number of engineering species for preparing skin tissue subcellular one in that third part, a primary method for the in vitro amplification of human epidermal cell 1 using a rotary bioreactor is use Cytodex-3 microcarrier The invention relates to a large-scale amplification human epidermal cell (hECs) of a culture system, which is a rotary type bioreactor container with high cross-section aspect ratio. The human epidermal cells are isolated from human skin by using a neutral protease and a trypsin-EDTA two-step method, culturing in a bioreactor (RCCS), and using an inverted microscope to attach the growth state of the cell to the microcarrier; the view of scanning electron microscope the cell population doubling time was analyzed to compare the difference of the in vitro proliferation of the microgravity culture and the plane culture. The main results were in the microgravity culture system of the rotary bioreactor, human epidermal cells can be quickly attached To the surface of the microcarrier, a large cell density is achieved during the culture, and a strong proliferative and cellular activity is shown. on the use of a rotary bioreactor and a microcarrier suspension culture epidermal cells, It is an effective method for preparing the skin tissue engineering seed cells in a large amount. The fourth part of the experiment uses a rotary bioreactor to prepare the particulate skin. 1. In vitro culture of human epidermal cells, the serum components in the culture solution are induced It can lead to a premature entry to a terminal state, and the proliferation tends to stop, usually for epidermal cells. In the early stage of culture, a serum-free culture medium (K-SFM) was used in the culture of human fibroblasts. interest. In view of the different requirements of the two cells for the culture solution, two different methods are tried in this experiment: the method of reducing the concentration gradient of the serum in the culture solution and the table The culture medium co-culture method, using Cytodex- 3 microcarriers and A rotary bioreactor vessel with high cross-section aspect ratio was used as a culture system to prepare the skin of the microparticles. The growth state of the cells was observed by scanning electron microscope. in that co-culture method of the main result, the micro-carrier particles have polygonal epidermal cells attached to the micro-carrier particle, the adhesion state of the fibroblasts is poor, microcarrier in that method of reduce the serum concentration gradient, the micro-carrier has a polygonal epidermal cell and a shuttle-shaped fibroblast, and the cell adhesion state is good, but the cell density is small.3 main conclusions are as follows:
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
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相關(guān)期刊論文 前5條
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