旋转式生物反应器内微载体培养扩增皮肤相关细胞及微粒皮肤的初步制备

发布时间：2019-05-16 22:57

【摘要】：随着细胞培养技术的发展,对皮肤相关细胞的培养,特别是人表皮细胞的培养有了很大的进展。在适宜的培养条件下,单个细胞在培养中不断增殖,逐渐融合成片状,形成了类似皮肤表皮层的上皮组织。这使得在较短的时间里,用大量表皮细胞培育成小片状的上皮组织成为可能。临床工作中,外科医生和细胞生物学家们也大量培养自体表皮细胞并用来修复皮肤缺损的病人。表皮细胞培养技术也从使用含血清培养液和细胞滋养层的培养方式,到不含血清、无滋养层的培养方式。培养方法的发展也促进了临床应用,从最初的细胞悬液移植到自体异体细胞膜片移植,以及表皮细胞生物材料复合物移植。培养和移植方法的发展使人们在治疗大面积皮肤缺损方面看到了希望。细胞培养技术对研究动物细胞的结构、功能和分化过程中的作用非常重要。因此,将微载体培养技术应用到贴壁依赖型细胞的大量培养,使上述研究进入到了一个新的层次。在微载体培养环境中,通过在培养液中轻柔的转动或者搅拌,细胞在微球表面呈现单层或者复层生长,在这种无复杂结构的悬浮培养体系中,可获得2x10~8个/毫升的细胞量。 Cytodex微载体是GE Healthcare公司为大量培养各类动物细胞专门开发的微载体,其培养体系的容器可以从数毫升到数千升,非常适合大规模扩增细胞。这种微载体的表面对细胞生长过程中的贴附和伸展过程专门优化,它的直径和密度也非常适合悬浮状态大规模培养扩增各类细胞。构成微载体球体的基质材料生物稳定性很好,提供给细胞适宜的表面生长环境。透光的球体也很容易通过显微镜观察贴附之上的细胞。通过以往的广泛使用可以看出,cytodex十分适合作为微载体培养细胞。旋转式生物反应器是由一个水平轴旋转的培养容器和与其共轴的氧合器组成,当容器以水平轴开始旋转时,充满其中的培养液作为一个整体也发生旋转,这种方式使内容物受到极小的剪切力。培养体系中的细胞旋转时所受的离心力、重力和科氏力抵消,因此细胞能大体维持悬浮状态。培养体系中的细胞受到极小的机械力的同时获得大量的氧、营养物质的供应。细胞所需的气体通过硅橡胶膜微量而持续的交换,避免了旋转培养体系中出现大的气泡和涡流。在临床工作中,使用自体表皮,成纤维细胞快速有效的修复各类皮肤缺损对皮肤愈合有很大意义,而足够数量的种子细胞对构建组织工程皮肤非常重要。本研究尝试使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器作为培养系统,大规模扩增人真皮成纤维细胞和人表皮细胞,期待能为各类组织工程皮肤产品提供充足的种子细胞。第一部分实验成纤维细胞和表皮细胞的体外培养及鉴定 1主要方法获取新生儿包皮切除术后组织,人表皮细胞使用中性蛋白酶和胰蛋白酶两步消化法获取;人真皮成纤维细胞使用胰酶消化组织块贴壁法获取。待原代培养成功后,常规传代。免疫组化方法进行抗波形丝蛋白、抗角蛋白染色,显微镜下观察。 2主要结果倒置显微镜下观察细胞形态和生长情况。人表皮细胞呈圆形,细胞铺满培养瓶底,密度均匀。人真皮成纤维细胞呈长梭形,胞核饱满。免疫组化结果显示人真皮成纤维细胞抗波形丝蛋白染色阳性,人表皮细胞抗角蛋白染色为阳性。 3主要结论通过鉴定,本实验所获取以及体外培养扩增的细胞分别为人真皮成纤维细胞和人表皮细胞。第二部分实验旋转式生物反应器培养扩增人真皮成纤维细胞 1主要方法利用组织贴壁法从人皮肤中分离出人真皮成纤维细胞(hDFBs)进行体外培养,使用DIO标记细胞后结合微载体在旋转式生物反应器(RCCS)中培养,细胞贴附微载体的状况使用荧光显微镜,扫描电镜观测。并且检测细胞周期和分析细胞群体倍增时间来比较微重力培养与平面培养的体外增殖能力差异。 2主要结果在旋转式生物反应器的微重力培养体系中,人成纤维细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,并且表现出很强的增殖能力和细胞活性。 3主要结论利用生物反应器和微载体悬浮培养人皮肤成纤维细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。第三部分实验使用旋转式生物反应器体外扩增人表皮细胞 1主要方法使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器容器作为培养系统大规模扩增人表皮细胞(hECs)。用中性蛋白酶和胰蛋白酶-EDTA两步骤法从人皮肤中分离出人表皮细胞,使用DIL标记细胞后结合微载体后在旋转式生物反应器(RCCS)中培养,细胞贴附微载体的生长状态使用倒置显微镜,扫描电镜观测。并且分析细胞群体倍增时间来比较微重力培养与平面培养的体外增殖能力差异。 2主要结果在旋转式生物反应器的微重力培养体系中,人表皮细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,并且表现出很强的增殖能力和细胞活性。 3主要结论使用旋转式生物反应器和微载体悬浮培养人表皮细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。第四部分实验使用旋转式生物反应器制备微粒皮肤 1主要方法人表皮细胞的体外培养中,培养液中的血清成分有诱导分化作用,会使其过早的进入终末状态,增殖趋于停止,通常对表皮细胞的培养早期使用角质细胞无血清培养液(K-SFM )。而在人成纤维细胞在体外培养过程中,含血清的培养液对其增殖有利。考虑到两种细胞对培养液的不同要求,本实验分别尝试两种不同方法:培养液血清浓度梯度降低法和表皮培养液共培养法,使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器容器作为培养系统尝试制备微粒皮肤。并使用扫描电镜观察细胞的贴附生长状态。 2主要结果表皮培养液共培养法中,微载体颗粒上有多角形的表皮细胞贴附,成纤维细胞贴附状态较差,部分从微载体上脱落。血清浓度梯度降低法中,微载体上有多角形的表皮细胞和梭形的成纤维细胞贴附,细胞贴附状态较好,但细胞密度较小。 3主要结论使用旋转式生物反应器和微载体悬浮培养微粒皮肤,培养液的血清浓度对共培养的两种细胞影响较大,使用血清浓度梯度降低法的共培养体系效果较好。
[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:
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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