CORE--CELL CULTURE

核心--细胞培养

• 批准号: 6100485
• 负责人: MARY L WILLIAMS
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6100485
• 关键词: biomedical facility; human tissue; keratinocyte; skin; tissue /cell culture

The Cell Culture Core will be responsible for the establishment, maintenance, and passaging of human keratinocytes from foreskin and adult skin samples. The work required of the Cell Culture Core technicians is more labor-intensive than routine cell culture activities. In addition to routine immersion type cultures, they will prepare lifted cultures, including formulation of matrix and feeder cell coatings, as well as growth and passage of cells lifted to the air/medium interface. The tedious and meticulous care required to prepare lifted and submerged cultures results from the need to coat each Nylaflo membrane in a collagen solution, and transfer it individually to a Petri dish, then covering it with another 1 ml of the collagen solution. Upon gelation, and after a 24 hour rinse with sterile distilled water, cells are plated onto the membranes and grown to confluence (7-10 days). Upon confluence, each membrane has to be carefully removed (lifed with forceps) from the initial dish (avoiding loss of any collagen from the side of the dish) and transferred to a larger dish containing two stacked, glass-wool filters, pre-soaked with medium. Submerged cultures must also be lifted and transferred to new dishes (without filters). Since each experiemtn generally consists of from 40 to 60 dishes, this activity is extremely time-consuming. The Cell Culture Core will prepare delipidized sera by two different techniques. Delipidization and dialysis of serum is time-consuming, requiring precision density-gradient centrifugation in potassium bromide at 50,000 rpms for 36-48 hrs. After centrifugation, the serum is dialyzed, using ten changes of PBS over the next four days, then the serum is filtered through a 0.22 um Millipore filter. The butanol/disopropyl ether method of serum delipidization is also labor- intensive. This procedure requires the addition of known volumes of solvent, repeated vigorous shakings, aliquoting, centrifuging, removal of the organic phase, and repetitation of the entire procedure. The serum is then lyophilized, and resuspended in distilled water to the original volume, after which it is filtered just before each use. The Cell Culture Core will store and classify cell lines, prepare cell suspensions for investigators as needed and perform routine assays, such as DNA and protein determinations. Personnel of the Cell Culture Core will do all of the microdocumentation of cultures, as needed by investigators, including phase microphotography, cell viability quantitation, and staining for low magnification visualization of culture densities.

细胞培养中心将负责建立， 来自包皮和成人的人角质形成细胞的维持和传代 皮肤样本 要求细胞培养核心技术人员的工作是 比常规细胞培养活动更劳动密集。 此外 对于常规的浸没式培养，他们将准备提升培养物， 包括基质和饲养细胞涂层的配制，以及 细胞的生长和传代被提升到空气/培养基界面。 的 准备提升和浸没时需要繁琐而细致的护理 培养的结果是需要在一个特定的环境中包被每个Nylaflo膜。 胶原蛋白溶液，并将其单独转移到培养皿中，然后 用另外1 ml的胶原蛋白溶液覆盖它。 在凝胶化时， 在用无菌蒸馏水冲洗24小时后，将细胞铺板 在膜上生长至汇合（7-10天）。 汇合后， 每个膜都必须小心地从 初始培养皿（避免从培养皿侧面损失任何胶原蛋白） 转移到一个更大的盘子里， 过滤器，用介质预浸泡。 还必须将淹没的培养物 并转移到新的培养皿中（没有过滤器）。 因为每次经历 一般由40至60道菜组成，这一活动是非常 耗时。 细胞培养核心将通过两种不同的方法制备脱脂血清。 技术. 血清的脱脂和透析是耗时的， 需要在溴化钾中进行精确的密度梯度离心 在50，000 rpm下持续36-48小时。 离心后， 透析，在接下来的四天内使用PBS的十次变化，然后 血清通过0.22 μ mMillipore过滤器过滤。 的 丁醇/二异丙醚法血清去脂也是劳动力- 密集。 该程序需要添加已知体积的 溶剂、反复剧烈振摇、等分、浓缩、取出 的有机相，并重复整个过程。 的 然后将血清冻干，并重悬于蒸馏水中， 原始体积，之后在每次使用前过滤。 细胞培养核心将储存和分类细胞系， 根据需要暂停研究者的研究，并进行常规检测， 作为DNA和蛋白质测定。 细胞培养中心人员 将根据需要， 研究人员，包括相位显微摄影、细胞活力 定量和染色，用于培养物的低放大率可视化 密度