Computational and experimental modeling of cell function in response to 3D oxygen transport in vitro.

摘要 将再生医学推向概括人体组织在物理上的结构和功能-- 生物长度尺度和相关的细胞密度，必须制定策略以满足它们无情的新陈代谢 要求。为了有效和有效地使fi氧化功能工程组织，我们必须了解 氧分压的变化如何调节细胞的活力、增殖和表型。几十年的细胞研究 在低氧水平下孵育的培养物揭示了低氧反应的某些方面，以及许多关键的低氧反应。 洞察它的机制。另外，一系列令人惊讶的生物材料已经被开发出来，这些材料可以支持 细胞在3D环境中，并且可以在更大程度上重现自然细胞的形态和功能 2D文化。然而，随着再生医学等新兴领域试图将细胞整合到这些 材料，出现了新的问题，关于氧运输和低氧在指导 细胞群体的密度和功能。目前，我们缺乏一个全面的框架来描述和预测 判断在时空存在的情况下，细胞群体将如何随时间改变其密度和功能 不均匀的氧气梯度。我们需要将我们对细胞对缺氧的反应的知识扩展到3D和 我们需要讲解局部氧气对组织特异性fic细胞功能的影响。在这份提案中，我和一位超级- 生物材料、计算建模和肝脏生物学方面的专家移植团队将统一计算 缺氧反应的模型和工程化模型组织将氧运输与组织功能联系起来 3D。我们的fi编码将被合并到一个经过实验验证的模型中，该模型能够预测细胞数量- Ten的密度和功能随特定的氧梯度而变化。细胞对缺氧的反应将是 由细胞特性fic响应函数参数化，并与基于试剂的氧气传输方程集成 计算模型。我们将使用先进的体积成像和图像分割来fi测试参数 3D水凝胶中活性、增殖、缺氧和表型的细胞标记的生化分析 含有HepG2肝细胞，一种来自高度代谢组织的良好的defiNed模型细胞类型。我假设我们的 flow的闭环计算和实验工作将产生组织中细胞行为的可扩展模型 这一水平捕捉到了以前未曾研究过的对缺氧的功能反应。最后，要广泛支持file Phe- 在氧气梯度存在下生长的细胞的非典型景观，我们将使用RNA测序来绘制空间图 灌流水凝胶中轴向和径向氧气梯度上细胞表型的分带性。受控封装 可复制结构的水凝胶中的细胞将使我们能够评估基因表达的这些空间模式- Sion具有一定程度的实验控制性和重复性，超出了体内方法的能力。已被占用 总之，这些研究将提供关于细胞如何对局部氧梯度做出反应的基本见解 3D环境。对氧梯度引入的空间异质性的分析也有望启发 组织内细胞功能工程化区域的新范例。

Abstract To advance regenerative medicine towards recapitulating the structures and functions of human tissues at physi- ologic length scales and with relevant cell densities, strategies must be developed to meet their unrelenting metabolic demands. In order to effectively and efficiently oxygenate functional engineered tissues, we must understand how changes in oxygen tension modulate cell viability, proliferation, and phenotype. Decades of studying cell cultures incubated under low oxygen levels have unveiled some aspects of the hypoxic response and many key in- sights into its mechanism. Separately, an astonishing array of biomaterials have been developed which can support cells in 3D environments and can recapitulate native cell morphologies and functions to a much greater extent than 2D culture. However, as emerging areas such as regenerative medicine have sought to incorporate cells within these materials, new questions have emerged regarding the roles played by oxygen transport and hypoxia in directing the density and function of the cell populations. Currently, we lack a comprehensive framework to describe and pre- dict how cell populations will alter their densities and functions over time in the presence of spatiotemporally heterogeneous oxygen gradients. We need to extend our knowledge of cellular responses to hypoxia into 3D and we need to profile how tissue-specific cell functions are impacted by local oxygen cues. In this proposal, I and a sup- porting team of experts in biomaterials, computational modeling, and liver biology will unify computational models of hypoxic response with engineered model tissues to link oxygen transport with tissue function in 3D. Our findings will be incorporated into an experimentally validated model capable of predicting how cell popula- tions change in density and function in response to specified oxygen gradients. Cellular responses to hypoxia will be parameterized by cell-specific response functions and integrated with oxygen transport equations in an agent-based computational model. We will fit parameters using advanced volumetric imaging and image segmentation along with biochemical assays to map cellular markers of viability, proliferation, hypoxia, and phenotype within 3D hydrogels containing HepG2 liver cells, a well-defined model cell type from a highly metabolic tissue. I hypothesize that our closed-loop computational and experimental workflow will yield a scalable model of cell behavior at the tissue level which captures previously unstudied functional responses to hypoxia. Finally, to broadly profile the phe- notypic landscape of cells growing in the presence of oxygen gradients, we will use RNA sequencing to map spatial zonation of cell phenotypes along axial and radial oxygen gradients in perfused hydrogels. Controlled encapsulation of cells within hydrogels of reproducible architecture will enable us to evaluate these spatial patterns in gene expres- sion with a degree of experimental control and reproducibility beyond the capabilities of in vivo approaches. Taken together, these studies will provide fundamental insights into how cells respond to local oxygen gradients in 3D environments. Analysis of the spatial heterogeneity introduced by oxygen gradients is also expected to inspire new paradigms for engineering zones of cell function within tissues.