Automated Perfused Culture Process for Adult Stem Cells

描述（由申请人提供）：来源于骨髓（BM）的贴壁间充质细胞群具有在体外和体内分化为多种细胞谱系的能力。这些群体，被称为骨髓基质细胞（MSC），在临床应用中治疗非造血系统疾病的潜在用途正在密切调查。离体扩增被用于增加可用于细胞和基因治疗应用的多能MSC的数量。目前还没有培养这些细胞的标准化方法，但是已经发现平板接种和培养细胞密度对于维持这些细胞的多能性至关重要。Aastrom之前开发了一种专有的单程灌注（SPP）工艺，该工艺成功地扩增了BM干细胞，并证明了其在人体中的植入能力。该工艺技术已在自动化临床培养系统AastromReplicellTM细胞生产系统（ARS）中有效实施，该系统能够在没有预先选择或分步培养的情况下在单步过程中生产细胞。该系统的培养室，沿着操作系统软件，都可以修改以适用于其他细胞类型的生长。使用该平台技术，所提出的研究的总体目标是开发符合GMP的自动化SPP培养过程，用于产生大量适合临床使用的高功能MSC。这一过程将是可靠的和可重复的，SPP和封闭系统自动化的独特组合应该允许MSC生产在GMP条件下，以最小的操作。在I期，将在小规模培养中定义已知显著影响贴壁细胞增殖和分化的基本培养参数，以优化所产生的功能性MSC的数量。将通过流式细胞术、集落测定和分化测定来鉴定MSC。将定义塑料表面特性，包括粗糙度、图案化和组织培养处理。接下来，将检查几种无血清培养基配方和表面涂层。还将对中等灌注和氧合水平的影响进行初步研究。在第二阶段，在进一步优化之后，将使用定义的参数来修改ARS组件，并且将验证扩展过程以确认在自动SPP系统中的可行性

DESCRIPTION (provided by applicant): Adherent mesenchymal cell populations derived from bone marrow (BM) have the capability of differentiating into multiple cell lineages in vitro and in vivo. The potential use of these populations, referred to as marrow stromal cells (MSC), in clinical applications to treat nonhematopoietic disorders is being intensely investigated. Ex vivo expansion is being used to increase the number of multipotent MSC available for cell and gene therapy applications. There are currently no standardized methodologies for the culture of these cells, however plating and culturing cell densities have been found to be critical in maintaining the multipotentiality of these cells. Aastrom has previously developed a proprietary single-pass perfusion (SPP) process that has successfully expands BM stem cells with demonstrated engraftment capability in humans. This process technology has been effectively implemented in an automated clinical culture system, the AastromReplicell (tm) Cell Production System (ARS), which enables cells to be produced in a single step process without pre-selection or step-cultures. The culture chamber of this system, along with the operating system software, can both be modified to apply for the growth of other cell types. Using this platform technology, the overall goal of the proposed studies is to develop a GMP compliant automated SPP culture process for the generation of large numbers of highly functional MSC suitable for clinical use. This process will be reliable and reproducible, and the unique combination of SPP and closed system automation should allow MSC production under GMP conditions with minimal manipulation. In Phase I, basic culture parameters known to significantly effect adherent cell proliferation and differentiation will be defined in small-scale cultures to optimize the number of functional MSC produced. MSC will be identified by flow cytometry, colony assays, and differentiation assays. Plastic surface characteristics, including roughness, patterning, and tissue-culture treatment, will be defined. Next, several serum-free media formulations and surface coatings will be examined. Initial studies on the effects of medium perfusion and oxygenation levels will also be performed. In Phase II, after further optimization, defined parameters will be used to modify the ARS components, and the expansion process will be validated to confirm feasibility in the automated SPP system