Real-time Monitoring of Critical Quality Attributes of Bioprinted Constructs in Manufacturing of Engineered Tissues

生物打印是自动沉积生物分子（例如活细胞和相关生物材料）以形成 3D 异质结构的过程。已经开发了几种生物打印技术来制造功能性细胞和组织结构，用于工程组织和器官的应用。生物打印工艺参数会影响制造结构内封装细胞的生物质量属性。目前，对这些关键生物质量属性的评估必须通过对构建体进行需要染色和切片的破坏性测定来离线进行。这一缺点影响了生物打印工艺向工业实践的转化。该奖项支持基础研究，以使用实时和过程中表征技术来预测生​​物打印结构的质量属性。研究结果将有助于增强美国的生物制造优势，特别是推进个性化医疗。在生物打印过程中，可以测量封装细胞响应不同频率交流电的电阻抗，并且阻抗信号中的任何扰动都会受到构建体的生物属性的影响。第一个研究目标是确定基准生物打印细胞结构的工艺参数（喷嘴直径、挤出压力和水凝胶浓度）与关键生物质量属性（细胞活力、密度和代谢状态）之间的相互作用。为了实现这一目标，将使用介电探针测量在交变电场（100 KHz 至 20 MHz）影响下印刷结构的介电常数。将分析获得的介电常数曲线，以提取与生物打印工艺参数相关的β色散属性（临界频率、电容下降和电容变化率）。 第二个目标是了解在施加电场下的生物打印过程中封装细胞的细胞膜极化改变的机制。为了实现这一目标，将开发生物打印过程的多物理模型，结合封装细胞的介电特性、生物材料特性和细胞动力学。该模型将通过最先进的离线表征方法进行实验校准。然后它将用于预测电场影响下的细胞极化状态。模拟结果将通过测量打印结构的生物质量属性来验证。第三个目标是确定生物打印工艺参数对哺乳动物和细菌细胞介电特性的影响。提取的介电常数属性将用于实时预测构建体的生物质量状态。这些预测将通过离线构建表征方法得到证实。

Bioprinting is the process of automated deposition of biological molecules, such as living cells and associated biomaterials, to form a 3D heterogeneous construct. Several bioprinting techniques have been developed to fabricate functional cellular and tissue constructs for applications in engineered tissues and organs. Bioprinting process parameters can affect biological quality attributes of encapsulated cells within fabricated constructs. Currently, assessment of these critical biological quality attributes must be performed offline by subjecting the constructs to destructive assays that require staining and sectioning. This drawback affects the translation of bioprinting processes to industrial practice. This award supports fundamental research to enable predicting the quality attributes of the bioprinted constructs using a real-time and in-process characterization technique. Research results will contribute to US biomanufacturing strengths, particularly towards advancing personalized medicine.In a bioprinting process, the electrical impedance of encapsulated cells in response to an alternating current at different frequencies can be measured, and any perturbations in the impedance signals are affected by the biological attributes of the constructs. The first research objective is to determine interactions between process parameters (nozzle diameter, extrusion pressure, and hydrogel concentration) and critical biological quality attributes (cell viability, density, and metabolic state) of benchmark bioprinted cellular constructs. To achieve this objective, the permittivity of printed constructs under the influence of an alternating electric field (100 KHz to 20 MHz) will be measured by using a dielectric probe. The obtained permittivity curves will be analyzed to extract beta-dispersion attributes (critical frequency, capacitance drop, and rate of capacitance change) which will be correlated to the bioprinting process parameters. The second objective is to understand the mechanism responsible for alterations in cellular membrane polarization of encapsulated cells in a bioprinting process under an applied electric field. To achieve this objective, a multi-physics model of the bioprinting process will be developed, combining the dielectric properties of encapsulated cells, biomaterial properties, and cellular kinetics. The model will be experimentally calibrated through state-of-the-art offline characterization methods. It then will be used to predict the cellular polarization states under the influence of an electric field. Simulations results will be verified by measuring the biological quality attributes of the printed constructs. The third objective is to determine the effects of bioprinting process parameters on the dielectric properties of mammalian and bacterial cells. The extracted permittivity attributes will be used to predict the biological quality state of the constructs in real-time. These predictions will be confirmed by offline construct characterization methods.