Igniting Life with Sparks of Light: 3D Spatiotemporal Photoactivation of Angiogenesis via Radiational Kinesis (3D SPARK)

复制人体器官在结构和功能上都是一个高度复杂的挑战。这一重大挑战的核心在于对血管化的迫切需求，以及更广泛地对细胞化的需求。我们身体中的细胞系统自然地以自下而上的方式编程，其中结构是功能的进化结果。例如，对最佳交换和运输的需要驱动形态发生，通过血管化、肺泡化和所有自组织隔室形成期间的动态信号传导和分泌模式来表现。组织工程师们已经尝试了反向希望功能也将遵循形式，并将重点放在结构问题上：产生无细胞结构的能力，例如用于运输的可灌注网络和用于细胞聚集的微孔支架。这些自上而下的工程基质是复杂的，但静态和非响应性的，给我们留下了大量播种，细胞化和刺激的基本手段，并限制了细胞介导的自下而上的生长和重塑。器官型生长模式是对生理需求的动态响应，由生化因子和刺激的时空控制释放驱动，并且需要能够自下而上重塑的极其柔软和可降解的细胞包封的细胞外微环境，这两种技术目前都只能在小微流体足迹中提供。3D SPARK项目为大型微流体提供了一个改变游戏规则的解决方案，通过计算机轴向光刻（CAL）和计算机轴向刺激（CAS）-计算机轴向断层扫描（CAT）的光学逆-进行的大规模体积组织生产。体积处理挑战了组织工程中的传统智慧，表明可以一次性产生复杂和精细的3D细胞结构，而不依赖于生物物质的缓慢，顺序处理，并且可以在单个细胞水平上访问大量制造的组织，而不需要物理操作或缓慢的光学扫描。在其核心，这种革命性的CAT启发的方法利用叠加的2D角光投影，以构建一个三维空间分布的曝光剂量，并体积触发光聚合（生物打印），光释放在光活性细胞包封的水凝胶基质中，通过光刺激（生物调节）和光激发（成像）来调节和监测组织发育期间的关键细胞事件。介导的体积处理和在多个波长下在3D中图案化光强度的能力，我们引入了一种可扩展的解决方案：（1）在这种软（<10 kPa）细胞包封的光活性凝胶中触发光聚合并制造完整的血管结构;（2）控制光诱导的化学物质（例如氧气）消耗（通过自由基淬灭）和分泌生化因子如生长因子（通过撑开）在整个体积上引导组织发育;以及（3）快速成像整个体积以监测与光调节和组织生长同时发生的3D细胞化。在我们的组织模型中，大血管网络等较大的特征以自上而下的方式设计和体积打印，并在内部涂覆内皮细胞（EC）。然后用光刺激诸如微血管毛细血管的更精细的特征，以从打印的凝胶内的稀疏封装的EC出现和发展，从而以自下而上的方式桥接大血管间隙。这个一体化平台超越了对基质的物理和化学性质进行图案化，从而能够动态操纵细胞过程，使我们能够同时适应自上而下的工程和自下而上的开发。因此，所提出的技术将是组织工程师的梦想工具，使他们能够以前所未有的方式在单细胞水平上利用光时空访问大量打印组织。

Replicating a human organ is a highly complex challenge both structurally and functionally. At the core of this grand challenge lies the critical need for vascularisation and more broadly the need for cellularisation. Cellular systems in our bodies are naturally programmed in a bottom-up fashion where structure is an evolutionary consequence of function. For instance, the need for optimal exchange and transport drives morphogenesis, manifesting itself via dynamic signalling and secretion patterns during vascularisation, alveolarization and the formation of all self-organised tissue compartments. Tissue engineers have attempted the inverse hoping function will also follow form, with a laser focus on the structure problem: the ability to produce acellular architectures such as perfusable networks for transport and microporous scaffolds for cellular aggregation. These top-down engineered matrices are intricate yet static and non-responsive, leaving us with rudimental means of bulk seeding, cellularisation and stimulation, and limiting cell-mediated bottom-up growth and remodelling. Organotypic growth patterns are a dynamic response to physiological needs, driven by the spatiotemporally controlled release of biochemical factors and stimuli, and require extremely soft and degradable cell encapsulated extracellular microenvironments capable of bottom-up remodelling, both of which are currently only afforded at small microfluidic footprints.The 3D SPARK project offers a game-changing solution to large-scale volumetric tissue production via computed axial lithography (CAL) and computed axial stimulation (CAS) - the optical inverses of computed axial tomography (CAT). Volumetric processing challenges conventional wisdom in tissue engineering showing that complex and delicate 3D cellular architectures can be produced all-at-once without relying on slow, sequential processing of biological matter, and that large volumes of manufactured tissue can be accessible at a single cell level without a need for physical manipulation or slow optical scanning. At its core, this revolutionary CAT-inspired method utilises a superposition of 2D angular light projections to construct a 3D spatial distribution of exposure dose, and volumetrically trigger photopolymerization (bioprinting), photorelease (biomodulation) and photoexcitation (imaging) to regulate and monitor key cellular events during tissue development in a photoactive cell-encapsulated hydrogel matrix.With light-mediated volumetric processing and the ability to pattern light intensity in 3D at multiple wavelengths, we introduce a scalable solution to: (1) trigger photopolymerization and manufacture intact vascular structures in such soft (<10 kPa) cell-encapsulated photoactive gels; (2) control the light-induced depletion of chemical species such as oxygen (via radical quenching), and secretion of biochemical factors such as growth factors (via uncaging) directing tissue development across the entire volume; and (3) rapidly image the entire volume to monitor 3D cellularisation concurrent with photomodulation and tissue growth. In our tissue models, larger features such as macrovascular networks are designed and volumetrically printed in a top-down fashion and are internally coated with endothelial cells (ECs). Finer features such as microvascular capillaries are then stimulated with light to emerge and develop from sparsely encapsulated ECs within the printed gel to bridge the macrovascular gaps in a bottom-up fashion. This all-in-one platform goes beyond patterning the physical and chemical properties of the matrix, to enable dynamic manipulation of cellular processes allowing us to accommodate for top-down engineering and bottom-up development simultaneously. Hence, the proposed technology will be the dream tool of tissue engineers giving them spatiotemporal access to large volumes of printed tissue at a single cell level with light in a way never achievable before.