通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印
Summary
这里介绍的是一种温和的3D打印技术，由交替的粘性惯性力驱动，以实现水凝胶微载体的构建。自制喷嘴具有灵活性，可轻松更换不同的材料和直径。可以获得直径为50-500μm的细胞结合微载体并收集以进行进一步培养。
Abstract
微载体是直径为60-250μm且比表面积较大的微球，通常用作大规模细胞培养的载体。微载体培养技术已成为细胞学研究的主要技术之一，在大规模细胞扩增领域得到普遍应用。微载体也被证明 在体外 组织工程构建和临床药物筛选中发挥着越来越重要的作用。目前制备微载体的方法包括微流控芯片和喷墨打印，它们通常依赖于复杂的流道设计，不兼容的两相界面和固定的喷嘴形状。这些方法面临着复杂的喷嘴加工，不方便的喷嘴更换以及应用于多个生物墨水时挤出力过大的挑战。在这项研究中，应用了一种称为交替粘性 – 惯性力喷射的3D打印技术，以构建直径为100-300μm的水凝胶微载体。随后将细胞接种在微载体上以形成组织工程模块。与现有方法相比，该方法为各种生物活性材料提供了自由的喷嘴尖端直径，灵活的喷嘴切换，自由控制打印参数和温和的打印条件。
Introduction
微载体是直径为60-250μm，比表面积较大的微球，通常用于细胞的大规模培养1，2。它们的外表面为细胞提供了丰富的生长位点，内部为空间增殖提供了支撑结构。球形结构还为监测和控制参数提供了便利，包括pH，O2以及营养物质和代谢物的浓度。当与搅拌罐生物反应器结合使用时，与传统培养物相比，微载体可以在相对较小的体积内实现更高的细胞密度，从而为实现大规模培养提供了一种经济有效的方法3。微载体培养技术已成为细胞学研究的主要技术之一，在干细胞、肝细胞、软骨细胞、成纤维细胞和其他结构的大规模扩增领域取得了很大进展4。它们也被发现是理想的药物输送载体和自下而上的单位，因此在临床药物筛选和 体外 组织工程修复中发挥着越来越重要的作用5。
为了满足不同场景下的力学性能要求，开发了多种类型的水凝胶材料，用于微载体的建造6，7，8，9，10，11。海藻酸盐和透明质酸（HA）水凝胶是两种最常用的微载体材料，因为它们具有良好的生物相容性和交联性12，13。海藻酸盐可以很容易地被氯化钙交联，其机械性能可以通过改变交联时间来调节。酪胺共轭HA通过过氧化氢和辣根过氧化物酶14催化的酪胺部分的氧化偶联而交联。胶原蛋白由于其独特的螺旋结构和交联纤维网络，经常被用作佐剂混合到微载体中，以进一步促进细胞附着15，16。
目前制备微载体的方法包括微流控芯片，喷墨打印和电喷雾17，18，19，20，21，22，23。微流控芯片已被证明在生产大小均匀的微载体方面具有快速高效的特性24。然而，该技术依赖于复杂的流道设计和制造工艺25。喷墨打印过程中的高温或过大的挤出力，以及电喷雾方法中的强电场，可能会对材料的性能产生不利影响，尤其是其生物活性19。此外，当应用于各种生物材料和直径时，这些方法中使用的定制喷嘴导致加工复杂性有限，成本高，灵活性低。
为了提供一种方便的微载体制备方法，应用了一种称为交替粘性惯性力喷射（AVIFJ）的3D打印技术来构建水凝胶微载体。该技术利用垂直振动过程中产生的向下驱动力和静压来克服喷嘴尖端的表面张力，从而形成液滴。在打印过程中，小的快速位移不是强烈的力和热条件，而是直接作用于喷嘴，对生物墨水的物理化学性质产生轻微影响，并对生物活性材料表现出极大的吸引力。利用AVIFJ方法，成功形成了直径为100-300 μm的多种生物材料的微载体。此外，微载体进一步证明可以很好地结合细胞，并为粘附的细胞提供合适的生长环境。
Protocol
Representative Results
Discussion
这里描述的方案为制备多种类型的水凝胶微载体和随后的细胞接种提供了说明。与微流控芯片和喷墨打印方法相比，AVIFJ构建微载体的方法具有更大的灵活性和生物相容性。独立的喷嘴使各种轻质喷嘴（包括玻璃微量移液器）可用于这些印刷系统。高度可控的加工使储液器体积、内径和打印头形状等参数可以自由调整。此外，一次性喷嘴便于在多种材料之间切换进行灭菌，从而避免了重复使用的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由北京市自然科学基金（3212007）、清华大学计划科研计划（20197050024）、清华大学春风基金（20201080760）、国家自然科学基金（51805294）、国家重点研发计划（2018YFA0703004）和111工程（B17026）共同资助。
Materials
| A549 cells | ATCC | CCL-185 | Human non-small cell lung cancer cell line |
| Bright field microscope | Olympus | DP70 | |
| Confocal microscope | Nikon | TI-FL | |
| Fetal bovine serum, FBS | BI | 04-001-1ACS | |
| Gelatin | SIGMA | G1890 | |
| Glass micropipettes | sutter instrument | b150-110-10 | |
| GlutaMAX | GIBCO | 35050-061 | |
| H-DMEM | GIBCO | 11960-044 | Dulbecco's modified eagle medium |
| Horseradish peroxidase powder | SIGMA | P6782 | |
| Hydrophobic agent | 3M | PN7026 | Follow the manufacturer's instructions and use after dilution |
| Micro-forge device | narishige | MF-900 | |
| Non-essential amino acids, NEAA | GIBCO | 11140-050 | non-essential amino acids |
| Penicillin G and streptomycin | GIBCO | 15140-122 | |
| Petri dish | SIGMA | P5731-500EA | |
| Puller | sutter instrument | P-1000 | |
| Sodium alginate | SIGMA | A0682 | |
| Trypsin | GIBCO | 25200-056 | |
| Type I collagen solution from rat tail | SIGMA | C3867 |
References
- Chen, A. K., Reuveny, S., Oh, S. K. W. Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: Achievements and future direction. Biotechnol Advances. 31, 1032-1046 (2013).
- Li, B., et al. Past, present, and future of microcarrier-based tissue engineering. Journal of Orthopaedic Translation. 3, 51-57 (2015).
- Badenes, S. M., Fernandes, T. G., Rodrigues, C. A. V., Diogo, M. M., Cabral, J. M. S. Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems. Journal of Biotechnol. 234, 71-82 (2016).
- de Soure, A. M., Fernandes-Platzgummer, A., Da Silva, C. L., Cabral, J. M. S. Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells. Journal of Biotechnol. 236, 88-109 (2016).
- Naqvi, S. M., et al. Living cell factories – electrosprayed microcapsules and microcarriers for minimally invasive delivery. Advanced Materials. 28, 5662-5671 (2016).
- Sarkar, S., et al. Chitosan: A promising therapeutic agent and effective drug delivery system in managing diabetes mellitus. Carbohydrate Polymers. 247, (2020).
- Sulaiman, S. B., Idrus, R. B. H., Hwei, N. M. Gelatin microsphere for cartilage tissue engineering: current and future strategies. Polymers. 12, (2020).
- Huang, L., Abdalla, A. M. E., Xiao, L., Yang, G. Biopolymer-based microcarriers for three-dimensional cell culture and engineered tissue formation. International Journal of Molecular Sciences. 21, (2020).
- Isiklan, N., Tokmak, S. Development of thermo/pH-responsive chitosan coated pectin-graft-poly(N, N-diethyl acrylamide) microcarriers. Carbohydrate Polymers. 218, 112-125 (2019).
- Lau, T. T., Wang, C., Wang, D. A. Cell delivery with genipin crosslinked gelatin microspheres in hydrogel/microcarrier composite. Composites Science & Technology. 70, 1909-1914 (2010).
- Lau, T. T. Hydrogel-microcarrier composite systems for cell delivery in tissue engineering. Acta Biomaterialia. 10, 1646-1662 (2014).
- Kwon, Y. J., Peng, C. A. Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture. Biotechniques. 33, 218 (2002).
- Leach, J. B., Bivens, K. A., Patrick, C. W., Schmidt, C. E. Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds. Biotechnology & Bioengineering. 82, 578-589 (2003).
- Kurisawa, M., Chung, J. E., Yang, Y. Y., Gao, S. J., Uyama, H. Injectable biodegradable hydrogels composed of hyaluronic acid-tyramine conjugates for drug delivery and tissue engineering. Chemical Communications. 34, 4312-4314 (2005).
- Yao, R., Alkhawtani, A. Y. F., Chen, R., Luan, J., Xu, M. Rapid and efficient in vivo angiogenesis directed by electro-assisted bioprinting of alginate/collagen microspheres with human umbilical vein endothelial cell coating layer. International Journal of Bioprinting. 5, 194 (2019).
- Mahou, R., Vlahos, A. E., Shulman, A., Sefton, M. V. Interpenetrating alginate-collagen polymer network microspheres for modular tissue engineering. Acs Biomaterials Science & Engineering. 4 (11), 3704-3712 (2017).
- Aftab, A., et al. Microfluidic platform for encapsulation of plant extract in chitosan microcarriers embedding silver nanoparticles for breast cancer cells. Applied Nanoscience. 10, 2281-2293 (2020).
- Park, W., et al. Microfluidic-printed microcarrier for in vitro expansion of adherent stem cells in 3D culture platform. Macromolecular Bioscience. 19, (2019).
- Chui, C., et al. Electrosprayed genipin cross-linked alginate-chitosan microcarriers for ex vivo expansion of mesenchymal stem cells. Journal of Biomedical Materials Research Part A. 107, 122-133 (2019).
- Min, N. G., Ku, M., Yang, J., Kim, S. Microfluidic production of uniform microcarriers with multicompartments through phase separation in emulsion drops. Chemistry of Materials. 28 (5), 1430-1438 (2016).
- Park, W., Jang, S., Kim, T. W., Bae, J., Lee, E. A. Microfluidic-printed microcarrier for in vitro expansion of adherent stem cells in 3D culture platform. Macromolecular Bioscience. 19, (2019).
- Xu, T., Kincaid, H., Atala, A., Yoo, J. J. High-Throughput Production of Single-Cell Microparticles Using an Inkjet Printing Technology. Journal of Manufacturing Science & Engineering. 130, 137-139 (2008).
- Rao, W., et al. Enhanced enrichment of prostate cancer stem-like cells with miniaturized 3D culture in liquid core-hydrogel shell microcapsules. Biomaterials. 27 (27), 7762-7773 (2014).
- Choi, C. H., Weitz, D. A., Lee, C. S. One step formation of controllable complex emulsions: From functional particles to simultaneous encapsulation of hydrophilic and hydrophobic agents into desired position. Advanced Materials. 25, 2536-2541 (2013).
- Choi, A., Seo, K. D., Kim, D. W., Kim, B. C., Dong, S. K. Recent advances in engineering microparticles and their nascent utilization in biomedical delivery and diagnostic applications. Lab On A Chip. 17 (4), 591-613 (2017).
- Liu, T., Pang, Y., Zhou, Z., Yao, R., Sun, W. An integrated cell printing system for the construction of heterogeneous tissue models. Acta Biomaterialia. 95, 245-257 (2019).
- Hassan, K., et al. Functional inks and extrusion-based 3D printing of 2D materials: a review of current research and applications. NANOSCALE. 12, 19007-19042 (2020).
- Vithani, K., et al. An overview of 3D printing technologies for soft materials and potential opportunities for lipid-based drug delivery systems. Pharmaceutical Research. 36, (2019).
