Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, Maryland, USA.
J Biomed Mater Res A. 2023 Aug;111(8):1279-1291. doi: 10.1002/jbm.a.37528. Epub 2023 Mar 14.
In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.
在组织工程领域，通常将 3D 支架和细胞结合起来，以生成用于治疗的构建体，从而修复或恢复患者的组织功能。为了实现治疗的预期作用机制，通常需要使用有活力的细胞，其中活细胞可以构建新组织，或者可以释放诱导组织再生的因子。因此，需要可靠地测量 3D 支架中的细胞活力，作为组织工程医疗产品的质量属性。在这里，我们开发了一种非侵入性、无标记的 3D 光学相干断层扫描 (OCT) 方法，可以快速（2.5 分钟）对大样本体积（1 毫米）进行成像，以评估支架内细胞活力和分布。使用由多糖基水凝胶接种人 Jurkat 细胞组成的模型支架-细胞系统评估 OCT 成像。使用了四个测试系统：接种活细胞的水凝胶、热休克或固定死细胞的水凝胶以及没有任何细胞的水凝胶。时间序列 OCT 图像显示，由于活细胞内折射率 (RI) 变化导致的散斑图案随时间的变化，而在纯水凝胶样品或死细胞的水凝胶中未观察到这种变化。散斑图案的变化用于通过图像相减生成活细胞对比度。通过这种方式，将 RI 变化较大的物体归类为活细胞。使用这种方法，OCT 成像测量平均每 0.288 毫米计算出 288 个活细胞中的 326 ± 52 个活细胞（在将细胞接种到凝胶中之前，通过吖啶橙-碘化丙啶细胞计数法预先确定）。考虑到制造支架-细胞构建体的过程中存在很大的不确定性，例如移液和细胞计数的误差，活细胞计数的 13%差异是合理的。此外，还绘制了活细胞在水凝胶支架内的 3D 分布，以评估其在整个体积内分布的均匀性。我们的结果表明，这是一种实时、非侵入性的方法，可以快速评估 3D 支架内活细胞的空间分布，这对于评估组织工程医疗产品可能很有用。
