三维球体间充质干细胞培养技术的研究进展及其应用

doi:10.3877/cma.j.issn.2095-1221.2023.04.005

三维（3D）培养是近年发展起来的一种新细胞培养技术，它能够模拟细胞在体内生长所需的微环境，让细胞与周围的细胞和微环境进行相互作用，发挥其独特的生物学功能。在培养3D间充质干细胞（MSCs）的过程中，选择合适的材料非常关键。目前，已有许多材料被应用于细胞增殖与分化的研究，包括天然材料、合成材料和生物材料。生物材料能够促进MSCs的黏附和增殖，而天然材料则能更好地模拟真实的MSCs生长环境。此外，3D-MSCs的体外培养包含有支架和无支架2种方式。支架培养适用于大规模的细胞培养，能够促进细胞的增殖和分化。在低氧环境下，3D-MSCs的线粒体呼吸和糖酵解活性增强，干性相关基因的表达也明显增加。因此，低氧环境对细胞有非常重要的影响，不仅改变细胞的代谢特性，还影响细胞的生理功能。本文对3D-MSCs培养的不同材料的应用及其优缺点、培养方式以及培养条件对细胞增殖和分化的影响进行综述。同时，本文探讨3D-MSCs在炎症性肠病、脊髓损伤、糖尿病和心肌梗死等方面的应用，旨在为未来的研究和临床应用提供方向。

关键词: 三维培养, 间充质干细胞, 培养材料, 培养方法, 应用进展

Three-dimensional (3D) culture is a contemporary cell culture technology that may imitate the microenvironment essential for cell growth in vivo, allowing cells to interact with neighboring cells and the microenvironment to exert their distinct biological roles. The selection of appropriate materials is critical in the culture of 3D-mesenchymal stem cells (MSCs) . Many materials, including natural, synthetic, and biological materials, are currently being used in cell proliferation and differentiationr research. Biological materials can boost the adhesion and proliferation of MSCs, whereas natural materials can better replicate the development environments of MSCs. Furthermore, the in vitro cultivation of 3D-MSCs includes scaffolds and no scaffolds. Scaffold culture is appropriate for large-scale cell culture and has the potential to stimulate cell proliferation and differentiation. Under hypoxic conditions, mitochondrial respiration and glycolytic activities were elevated in 3D-MSCs, as was the expression of stemness-related genes. As a result, the hypoxic environment significantly impacts on cells, altering not just their metabolic features but also their physiological functions. This page discusses several materials for culturing 3D-MSCs, their benefits and drawbacks, culture methods, and the effects of culture conditions on cell proliferation and differentiation. At the same time, this paper discusses the use of 3D-MSCs in inflammatory bowel disease, spinal cord injury, diabetes, and myocardial infarction to provide directions for future research and clinical application and anticipating significant progress in the medical field.

Key words: Three-dimensional culture, Mesenchymal stem cells, Culture materials, Culture methods, Application progress

表1 常见的3D--MSCs培养材料

材料类型	种类	优势	劣势	参考文献
天然材料	胶原蛋白、明胶、透明质酸	促进细胞黏附；支架的孔隙率利于细胞增殖	材料可塑性差；材料价格偏高	[12,13,14]
合成材料	聚乳酸、聚乙醇酸、聚乳酸乙醇胺	材料的可塑性强；良好的生物力学性能	细胞毒性；材料相容性较差	[15,16,17]
生物材料	水凝胶、壳聚糖、琼脂糖	良好生物相容性；材料易降解	材料可塑性差	[18,19,20]

表1 常见的3D--MSCs培养材料

材料类型	种类	优势	劣势	参考文献
天然材料	胶原蛋白、明胶、透明质酸	促进细胞黏附；支架的孔隙率利于细胞增殖	材料可塑性差；材料价格偏高	[12,13,14]
合成材料	聚乳酸、聚乙醇酸、聚乳酸乙醇胺	材料的可塑性强；良好的生物力学性能	细胞毒性；材料相容性较差	[15,16,17]
生物材料	水凝胶、壳聚糖、琼脂糖	良好生物相容性；材料易降解	材料可塑性差	[18,19,20]

表2 比较3D-MSCs两种支架培养方法

培养方法	种类	优势	劣势	参考文献
无支架	悬滴法；旋转培养；微孔板	操作简单；可提高细胞培养的效率和准确性	空间受限，不利于大规模生产；速度较慢，需长时间才能得到足够数量的细胞；环境难以控制，易受到外界因素的影响生长	[21,22,23]
有支架	水凝胶；微载体；生物聚合物	可更好地提供支撑作用，促进细胞生长；可控制细胞生长的环境；可实现大规模生产	制备和处理过程复杂，可能会对细胞产生不良影响；成本较高	[24,25,26]

表2 比较3D-MSCs两种支架培养方法

培养方法	种类	优势	劣势	参考文献
无支架	悬滴法；旋转培养；微孔板	操作简单；可提高细胞培养的效率和准确性	空间受限，不利于大规模生产；速度较慢，需长时间才能得到足够数量的细胞；环境难以控制，易受到外界因素的影响生长	[21,22,23]
有支架	水凝胶；微载体；生物聚合物	可更好地提供支撑作用，促进细胞生长；可控制细胞生长的环境；可实现大规模生产	制备和处理过程复杂，可能会对细胞产生不良影响；成本较高	[24,25,26]

图1 常见的3D--MSCs支架培养模型注：a图为3D--MSCs水凝胶培养模型；b图为3D--MSCs微载体培养模型；c图为3D--MSCs生物聚合物支架培养模型；d图为3D--MSCs生物反应器培养模型

1	Follin B, Juhl M, Cohen S, et al. Increased paracrine immunomodulatory potential of mesenchymal stromal cells in three-dimensional culture[J]. Tissue Eng Part B Rev, 2016, 22(4):322-329.
2	Kouroupis D, Correa D. Increased mesenchymal stem cell functionalization in three-dimensional manufacturing settings for enhanced therapeutic zpplications[J]. Front Bioeng Biotechnol, 2021, 9:621748.
3	Jauković A, Abadjieva D, Trivanović D, et al. Specificity of 3D MSC spheroids microenvironment: impact on MSC behavior and properties[J]. Stem Cell Rev Rep, 2020, 16(5):853-875.
4	鲍颖颖,陈小湧.间充质干细胞防治慢性移植物抗宿主病:进展与挑战[J].器官移植, 2023, 14(3):327-335.
5	李芳,李栋,刘欢,等.三维培养对脐带间充质干细胞表型及造血因子基因表达的影响[J].中国实验血液学杂志, 2014, 22(5):1408-1414.
6	Sayin E, Baran ET, Elsheikh A, et al. Evaluating oxygen tensions related to bone marrow and matrix for MSC differentiation in 2D and 3D biomimetic lamellar scaffolds[J]. Int Mol Sci, 2021, 22(8):4010.
7	Kusuma GD, Li A, Zhu D, et al. Effect of 2D and 3D culture microenvironments on mesenchymal stem cell-derived extracellular vesicles potencies[J]. Front Cell Dev Biol, 2022, 10:819726.
8	Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells[J]. Cell Transplant, 2011, 20(1):5-14.
9	Levy O, Kuai R, Siren EMJ, et al. Shattering barriers toward clinically meaningful MSC therapies[J]. Sci Advs, 2020, 22:6(30):eaba6884.
10	Al-Shaibani MBH. Three-dimensional cell culture(3DCC)improves secretion of signaling molecules of mesenchymal stem cells(MSCs)[J]. Biotechnol Lett, 2022, 44(1):143-155.
11	Hoang DM, Pham PT, Bach TQ, et al. Stem cell-based therapy for human diseases[J]. Signal Transduct Target Ther, 2022, 7(1):272.
12	Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture[J]. Nature methods, 2016,13(5):405-414.
13	Yap JX, Leo CP, Mohd Yasin NH, et al. Recent advances of natural biopolymeric culture scaffold: synthesis and modification[J]. Bioengineered, 2022, 13(2):2226-2247.
14	Zhai P, Peng X, Li B, et al. The application of hyaluronic acid in bone regeneration[J]. Int Biol Macromol, 2020, 151:1224-1239.
15	Hunt NC, Hallam D, Karimi A, et al. 3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development[J]. Acta Biomater, 2017, 49:329-343.
16	Wasyłeczko M, Sikorska W, Chwojnowski A. Review of synthetic and hybrid scaffolds in cartilage tissue engineering[J]. Membranes (Basel), 2020,10(11):348.
17	Rodina AV, Tenchurin TK, Saprykin VP, et al. Proliferative and differentiation potential of multipotent mesenchymal stem cells cultured on biocompatible polymer scaffolds with various physicochemical characteristics[J]. Bull Exp Biol Med, 2017, 162(4):488-495.
18	Sun W, Gregory DA, Tomeh MA, et al. Silk fibroin as a functional biomaterial for tissue engineering[J]. Int Mol Sci, 2021, 22(3):1499.
19	Lemoine M, Casey SM, OByrne JM, et al. The development of natural polymer scaffold-based therapeutics for osteochondral repair[J]. Biochem Soc Trans, 2020, 48(4):1433-1445.
20	Ullah F, Javed F, Zakaria MR, et al. Determining the molecular-weight and interfacial properties of chitosan built nanohydrogel for controlled drug delivery applications[J]. Biointerface Res Appl Chem, 2019, 4452-4457.
21	Kampleitner C, Changi K, Felfel RM, et al. Preclinical biological and physicochemical evaluation of two-photon engineered 3D biomimetic copolymer scaffolds for bone healing[J]. Biomater Sci, 2020, 8(6):1683-1694.
22	梁婷婷,张世昌.间充质干细胞球形体培养的研究进展[J/OL].中华细胞与干细胞杂志（电子版）, 2021,11(6):372-377.
23	Costa EC, de Melo-Diogo D, Moreira AF, et al. Spheroids formation on non-adhesive surfaces by liquid overlay technique: considerations and practical approaches[J]. Biotechnol J, 2018 ,13(1). doi: 10.1002/biot.201700417.
24	张斌斌,高全文,李冰,等.骨髓间充质干细胞在Matrigel凝胶支架上的生长与变化[J].中国组织工程研究, 2018, 22(13):1993-1998.
25	Chen Q, Wang Y. The application of three-dimensional cell culture in clinical medicine[J]. Biotechnol Lett, 2020,42(11):2071-2082.
26	Alghuwainem A, Alshareeda AT, Alsowayan B. Scaffold-free 3-D cell sheet technique bridges the Gap between 2-D cell culture and animal models[J]. Int J Mol Sci, 2019 , 20(19):4926.
27	Ezquerra S, Zuleta A, Arancibia R, et al.Functional properties of human-derived mesenchymal stem cell spheroids: a meta-analysis and systematic review[J]. Stem Cells Int, 2021:8825332.
28	Falcones B, Sanz-Fraile H, Marhuenda E, et al. Bioprintable Lung extracellular matrix hydrogel scaffolds for 3D culture of mesenchymal stromal cells[J]. Polymers (Basel), 2021, 13(14):2350.
29	Lamparelli EP, Lovecchio J, Ciardulli MC, et al. Chondrogenic commitment of human bone marrow mesenchymal stem cells in a perfused collagen hydrogel functionalized with hTGF-β1-releasing PLGA microcarrier[J]. Pharmaceutics, 2021, 13(3):399.
30	Chen CY, Li C, Ke CJ, et al. Kartogenin enhances chondrogenic differentiation of MSCs in 3D Tri-copolymer scaffolds and the self-designed bioreactor system[J]. Biomolecules, 2021,11(1):115.
31	Cao J, Wang B, Tang T, et al. Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury[J]. Stem Cell Res Ther, 2020, 11(1):206.
32	Rybkowska P, Radoszkiewicz K, Kawalec M, et al. The metabolic changes between monolayer (2D) and three-dimensional (3D) culture conditions in human mesenchymal stem/stromal cells derived from adipose tissue[J]. Cells, 2023,12(1):178.
33	Wuchter P, Diehlmann A, Klüter H. Closer to Nature: The role of MSCs in recreating the microenvironment of the hematopoietic stem cell Niche in vitro[J]. Transfus Med Hemother,2022,49(4):258-267.
34	Theodoridis K, Aggelidou E, Manthou ME, et al. Hypoxia promotes cartilage regeneration in cell-seeded 3D-printed bioscaffolds cultured with a bespoke 3D culture device[J]. Int J MolSci, 2023,24(7):6040.
35	Silva-Carvalho Aé, da Silva IGM, Corrêa JR, et al. Regulatory T-cell enhancement, expression of adhesion molecules, and production of anti-inflammatory factors are differentially modulated by spheroid-cultured mesenchymal stem cells[J]. Int J MolSci, 2022,23(22):14349.
36	Li Q, Lei X, Wang X, et al. Hydroxyapatite/collagen three-dimensional printed scaffolds and their osteogenic effects on human bone marrow-derived mesenchymal stem cells[J]. Tissue Eng Part A, 2019, 25(17-18):1261-1271.
37	Fernández-Francos S, Eiro N, González-Galiano N, et al . Mesenchymal stem cell-based therapy as an alternative to the treatment of acute respiratory distress syndrome: current evidence and future perspectives[J]. Int Mol Sci, 2021, 22(15):7850.
38	Huldani H, Margiana R, Ahmad F, et al. Immunotherapy of inflammatory bowel disease (IBD) through mesenchymal stem cells[J]. Int Immunopharmacol, 2022, 107:108698.doi: 10.1016/j.intimp.2022.108698.
39	Wang Y, Huang B, Jin T, et al. Intestinal fibrosis in inflammatory bowel disease and the prospects of mesenchymal stem cell therapy[J]. Front Immunol, 2022,13:835005.
40	Regmi S, Seo Y, Ahn JS, et al. Heterospheroid formation improves therapeutic efficacy of mesenchymal stem cells in murine colitis through immunomodulation and epithelial regeneration[J]. Biomaterials, 2021, 271:120752.
41	Gonzalez Pujana A, Beloqui A, Javier Aguirre J, et al. Mesenchymal stromal cells encapsulated in licensing hydrogels exert delocalized systemic protection against ulcerative colitis via subcutaneous xenotransplantation[J]. Eur Pharm Biopharm, 2022, 172:31-40.
42	Saadh MJ, Mikhailova MV, Rasoolzadegan S, et al. Therapeutic potential of mesenchymal stem/stromal cells (MSCs)-based cell therapy for inflammatory bowel diseases (IBD) therapy[J]. Eur J Med Res, 2023, 28(1):47.
43	Guo S, Perets N, Betzer O, et al. Intranasal delivery of mesenchymal stem cell derived exosomes loaded with phosphatase and tensin homolog siRNA repairs complete spinal cord injury[J]. ACS Nano, 2019, 13:10015-10028.
44	Saremi J, Mahmoodi N, Rasouli M, et al. Advanced approaches to regenerate spinal cord injury: the development of cell and tissue engineering therapy and combinational treatments [J]. Biomed Pharmacother, 2022,146:112529.
45	Liu T, Zhu W, Zhang X, et al. Recent advances in cell and functional biomaterial treatment for spinal cord injury[J]. Biomed Res Int, 2022, 2022:5079153.doi: 10.1155/2022/5079153.
46	Lv B, Zhang X, Yuan J, et al. Biomaterial-supported MSC transplantation enhances cell-cell communication for spinal cord injury[J]. Stem Cell Res Ther, 2021, 12(1):36.
47	Deng J, Li M, Meng F, et al. 3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice[J]. Cell Dea-th Dis,2021,12(12):1096.
48	Liu L, Wan J, Dai M, et al. Effects of oxygen generating scaffolds on cell survival and functional recovery following acute spinal cord injury in rats[J]. J Mater Sci Mater Med, 2020, 31(12):115.
49	Zhu L, Wang S, Qu J, et al. The therapeutic potential of mesenchymal stem cells in the treatment of diabetes mellitus[J]. Cell Reprogram, 2022, 24(6):329-342.
50	Gao S, Zhang Y, Liang K, et al. Mesenchymal stem cells (MSCs): a novel therapy for type 2 diabetes[J]. Stem Cells Int, 2022, 2022:8637493.doi: 10.1155/2022/8637493.
51	Ogurtsova K, Guariguata L, Barengo NC, et al. IDF diabetes Atlas: Global estimates of undiagnosed diabetes in adults for 2021[J]. Diabetes Res Clin Pract, 2022, 183:109118.
52	Barati G, Nadri S, Hajian R, etal. Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats[J]. Cell Physiol, 2019, 234(5):6801-6809.
53	Zhang Y, Gao S, Liang K, et al. Exendin-4 gene modification and microscaffold encapsulation promote self-persistence and antidiabetic activity of MSCs[J]. Sci Adv, 2021, 7(27):eabi4379.
54	Tang J, Cui X, Zhang Z, et al. Injection-free delivery of MSC-derived extracellular vesicles for myocardial infarction therapeutics[J]. Adv Healthc Mater, 2022, 11(5):e2100312.
55	Yamada Y, Minatoguchi S, Kanamori H, et al. Stem cell therapy for acute myocardial infarction focusing on the comparison between Muse cells and mesenchymal stem cells[J]. J Cardiol, 2022, 80(1):80-87.
56	Csöbönyeiová M, Beerová N, Klein M, et al. Cell-based and selected cell-free therapies for myocardial infarction: how do they compare to the current treatment options?[J]. Int J Mol Sci, 2022,23(18):10314.
57	Meng H, Cheng W, Wang L, et al. Mesenchymal stem cell exosomes in the treatment of myocardial infarction: a systematic review of preclinical in vivo studies[J]. Cardiovasc Transl Res, 2022,15(2):317-339.
58	Gao L, Yi M , Xing M , et al. In situ activated mesenchymal stem cell (MSCs) by bioactive hydrogel for myocardial infarction treatment[J]. J Mater Chem B, 2020, 8(34):7713-7722.
59	Qazi REM, Khan I, Haneef K, et al . Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model[J]. World J Stem Cells, 2022, 14(8):633-657.

[1]	沈纵, 魏晨如, 朱邦晖, 包郁露, 伍国胜, 孙瑜. 间充质干细胞治疗吸入性损伤的动物实验研究进展[J]. 中华损伤与修复杂志(电子版), 2023, 18(02): 180-183.
[2]	唐英俊, 李华娟, 王赛妮, 徐旺, 刘峰, 李羲, 郝新宝, 黄华萍. 人脐带间充质干细胞治疗COPD小鼠及机制分析[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 476-480.
[3]	李晔, 何洁, 胡锦秀, 王金祥, 田川, 潘杭, 陈梦蝶, 赵晓娟, 叶丽, 张敏, 潘兴华. 高活性间充质干细胞干预猕猴卵巢衰老的研究[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 210-219.
[4]	刘文慧, 吴涛, 张曦. 间充质干细胞联合血小板生成素受体激动剂在异基因造血干细胞移植后血小板恢复中的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 242-246.
[5]	王红敏, 谢云波, 王彦虎, 王福生. 间充质干细胞治疗新冠病毒感染的临床研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 247-256.
[6]	秦富豪, 郑正, 江滨. 间充质干细胞在克罗恩病肛瘘治疗中的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 172-177.
[7]	袁久莉, 刘丹, 李林藜, 刘晋宇. 毛囊间充质干细胞的基础研究及临床应用[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 189-192.
[8]	冯星, 靳洪涛, 马隽, 宋永周, 刘爱京. 间充质干细胞治疗炎性关节炎的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 87-92.
[9]	雷双银, 习剑鑫, 贺羽轩, 姚静宜, 石博雅, 马杰, 池光范, 李美英. 间充质干细胞源外泌体在神经退行性疾病治疗中的应用与进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 93-100.
[10]	符莞孟, 王晓黎, 刘玉, 张潍, 张菊. 干细胞治疗多囊卵巢综合征的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 108-114.
[11]	陈玉婷, 周影, 陆雅斐, 江滨. 缺氧预处理间充质干细胞的功能及机制研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 115-120.
[12]	杨蕴钊, 周诚, 石美涵, 赵静, 白雪源. 人羊水间充质干细胞对膜性肾病大鼠的治疗作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 181-186.
[13]	宋艳琪, 任雪景, 王文娟, 韩秋霞, 续玥, 庄凯婷, 肖拓, 蔡广研. 间充质干细胞对顺铂诱导的小鼠急性肾损伤中细胞铁死亡的作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 187-193.
[14]	陈客宏. 干细胞外泌体防治腹膜透析腹膜纤维化新技术研究[J]. 中华肾病研究电子杂志, 2023, 12(03): 180-180.
[15]	梁宇同, 丁旭, 马国慧, 黄艳红. 间充质干细胞在宫腔粘连治疗中的研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(05): 596-599.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Research progress and application of three-dimensional spherical mesenchymal stem cell culture technology

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Research progress and application of three-dimensional spherical mesenchymal stem cell culture technology