类器官的培养方法及应用前景

doi:10.16431/j.cnki.1671-7236.2023.07.010

摘要/Abstract

摘要： 近年来随着体外建模领域研究的不断深入，发现细胞试验存在着细胞单一、难以模拟机体复杂的组织结构和正常生理、病理状态等缺点，以及动物试验的昂贵费用和相关生理伦理等问题，类器官逐渐成为科学家们关注的热点。类器官来源于机体自身组织及干细胞，是通过体外3D培养形成的具有原始组织及器官的三维结构细胞团块，在组织结构、细胞类型和功能等方面与来源组织高度一致。这种类器官模型的构建，不仅为替代再生组织及器官的研究提供更多方法，还在组织移植和修复等工作中发挥重要作用。作者主要对促进类器官成熟的培养方法进行介绍，详细阐述了微孔阵列法、微流体法、生物反应器法和水凝胶基质法4种方法，并回顾了有关类器官培养系统的历史演变，分析了近年来学者们在类器官研究领域中存在的诸多限制和未来挑战，以及类器官在新兴工程技术中的应用前景和展望，旨在为类器官培养方法的进一步完善及在相关领域的研究和应用提供更多的理论依据和新的思路。

关键词: 类器官; 培养方法; 3D细胞培养; 应用现状

Abstract: In recent years, with the deepening of research in the field of in vitro modeling, it has been found that cell tests have some shortcomings, such as single cell, difficult to simulate complex tissue structure and normal physiological and pathological state of the body, and the high cost of animal tests and related physiological ethics and other issues, organoids have gradually become the focus of attention of scientists.Organoids are derived from the body's own tissues and stem cells, which are 3D structured cell clumps with original tissues and organs formed through 3D culture in vitro.They are highly consistent with the source tissues in terms of tissue structure, cell type and function.The construction of organoid model not only provides more methods for the research of replacing regenerated tissues and organs, but also plays an important role in tissue transplantation and repair.This review mainly introduces the culture methods for promoting organoid maturation, elaborates four methods, including microporous array method, microfluid method, bioreactor method and hydrogel matrix method, reviews the historical evolution of organoid culture system, and analyzes the limitations and future challenges in the field of organoid research in recent years.As well as the application prospect of organoids in emerging engineering technology, it aims to provide more theoretical basis and new ideas for further improvement of organoids culture methods and research and application in related fields.

Key words: organoids; cultural method; 3D cell culture; application status

中图分类号:

S858

王曼茜, 李庆豪, 孙娟, 胡慧, 金鑫. 类器官的培养方法及应用前景[J]. 中国畜牧兽医, 2023, 50(7): 2688-2696.

WANG Manxi, LI Qinghao, SUN Juan, HU Hui, JIN Xin. Culture Methods and Application Prospects of Organoids[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2688-2696.

参考文献

[1] KRATOCHVIL M J, SEYMOUR A J, LI T L, et al.Engineered materials for organoid systems[J].Nature Reviews Materials, 2019, 4(9):606-622.
[2] GARRETA E, KAMM R D, LANCASTER M A, et al.Rethinking organoid technology through bioengineering[J].Nature Materials, 2020, 20(2):147-155.
[3] SATO T, VRIES R G, SNIPPERTH J, et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[J].Nature Materials, 2009, 45(9):262-265.
[4] SPENCE J R, MAYHEW C N, RANKIN S A, et al.Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro[J].Nature, 2011, 470(7332):105-109.
[5] DEDHIA P H, BERTAUX S N, ZAVROS Y, et al.Organoid models of human gastrointestinal development and disease[J].Gastroenterology, 2016, 150(5):1098-1112.
[6] MCCRACKEN K W, CATÁ E M, CRAWFORD C M, et al.Modelling human development and disease in pluripotent stem-cell-derived gastric organoids[J].Nature Materials, 2014, 516(7531):400-404.
[7] NICK B, MERITXELL H, PEKKA K, et al.Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro[J].Cell Stem Cell, 2010, 6(1):25-36.
[8] SUGIMOTO S, OHTA Y, FUJII M, et al.Reconstruction of the human colon epithelium in vivo[J].Cell Stem Cell, 2018, 22(2):171-176.
[9] XIE B Y, WU A W.Organoid culture of isolated cells from patientderived tissues with colorectal cancer[J].Chinese Medical Journal, 2016, 129(20):2469-2475.
[10] SATO T, STANGE D E, MARC F, et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[J].Gastroenterology, 2011, 141(5):1762-1772.
[11] DYE B R, HILL D R, FERGUSON M A, et al.In vitro generation of human pluripotent stem cell derived lung organoids[J].eLife, 2015, 4:e05098.
[12] CHAPMAN H A, XIAOPENG L, ALEXANDER J P, et al.Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice[J].Journal of Clinical Investigation, 2011, 121(7):2855-2862.
[13] SHIN K, LEE J, GUO N, et al.Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder[J].European Urology, 2012, 61(6):1263-1264.
[14] LANCASTER M A, MAGDALENA R, CAROL A M, et al.Cerebral organoids model human brain development and microcephaly[J].Nature, 2013, 501(7467):373-379.
[15] LANCASTER M A, KNOBLICH J A.Generation of cerebral organoids from human pluripotent stem cells[J].Nature Protocols, 2014, 9(10):2329-2340.
[16] AU S H, CHAMBERLAIN M D, MAHESH S, et al.Hepatic organoids for microfluidic drug screening[J]. Lab on a Chip, 2014, 14(17):3290-3299.
[17] LEITE S B, ROOSENS T, TAGHDOUINI A E, et al.Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro[J].Biomaterials, 2016, 78:1-10.
[18] HUCH M, DORRELL C, BOJ S F, et al.In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration[J].Nature, 2013, 494(7436):247-250.
[19] TAKEBE T, SEKINE K, ENOMURA M, et al.Vascularized andfunctional human liver from an iPSC-derived organ bud transplant[J].Nature, 2013, 499(7459):481-484.
[20] BARKAUSKAS C E, CRONCE M J, RACKLEY C R, et al.Type 2 alveolar cells are stem cells in adult lung[J].Juurnal of Clinical Investigation, 2012, 123(7):3025-3036.
[21] HUCH M, BONFANTI P, BOJ S F, et al.Unlimited in vitro expansionof adult bi-potent pancreas progenitors through the Lgr5/R- spondin axis[J]. EMBO Journal, 2013, 32(20):2708-2721.
[22] BRACCINI A, WENDT D, JAQUIERY C, et al.Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts[J]. Cell Stem Cell, 2005, 23(8):1066-1072
[23] KIM G A, SPENCE J R, TAKAYAMA S.Bioengineering for intestinal organoidcultures[J].Current Opinion Biotechnology, 2017, 47:51-58.
[24] KANG S M, KIM D, LEE J H, et al.Engineered microsystemsfor spheroid and organoid studies[J].Advanced Healthcare Materials, 2021, 10(2):e2001284.
[25] VELASCO V, SHARIATI S A, ESFANDYARPOUR R.Microtechnology-based method for organoid models[J].Microsystems & Nanoengineering, 2020, 6:76.
[26] YIN X, MEAD B E, SAFAEE H, et al.Engineering stem cell organoids[J].Cell Stem Cell, 2016, 18(1):25-38.
[27] BRASSARD J A, LUTOLF M P.Engineering stem cell self-organization to buildbetter organoids[J].Cell Stem Cell, 2019, 24(6):860-876.
[28] VARZIDEH F, PAHLAVAN S, ANSARI H, et al.Human cardiomyocytes undergo nhanced maturation in embryonic stem cell-derived organoid transplants[J].Biomaterials, 2019, 192:537-550.
[29] 何懿, 栾杰.PDMS微孔阵列细胞培养实验平台的初步构建与评测[J].组织工程与重建外科杂志, 2011, 7(3):121-128. HE Y, LUAN J.Preliminary construction and evaluation of PDMS microarray cell culture experimental platform[J].Journal of Tissue Engineering and Reconstructive Surgery, 2011, 7(3):121-128.(in Chinese)
[30] MURROW L M, WEBER R J, GARTNER Z J.Dissecting the stem cell niche with organoid models:An engineering-based approach[J].Development, 2017, 144(6):998-1007.
[31] CHEN C, RENGARAJAN V, KJAR A, et al.A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays[J].Bioactive Materials, 2020, 6(4):1130-1139.
[32] BRANDENBERG N, HOEHNEL S, KUTTLER F, et al.High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays[J].Nature Biomedical Engineering, 2020, 4(9):863-874.
[33] TAKEBE T, SEKINE K, KIMURA M, et al.Massive and reproducible production of liver buds entirely from human pluripotent stem cells[J].Cell Reports, 2017, 21(10):2661-2670.
[34] ROSSEN N S, ANANDAKUMARAN P N, NIEDEN R Z, et al.Injectable therapeutic organoids using sacrificial hydrogels[J].iScience, 2020, 23(5):101052.
[35] 林路遥, 林金明.微流控芯片上细胞培养与分析方法研究进展[J].分析科学学报, 2017, 33(5):707-714. LIN L Y, LIN J M.Research progress of cell culture and analysis on microfluidic chip[J].Chinese Journal of Analytical Science, 2017, 33(5):707-714.(in Chinese)
[36] 陈序, 蔡博, 刘文宇, 等.微流控芯片在类器官技术中的应用[J].中华实验外科杂志, 2022, 39(6):1215-1221. CHEN X, CAI B, LIU W Y, et al.Application of microfluidic chip in organoid technology[J].Chinese Journal of Experimental Surgery, 2022, 39(6):1215-1221.(in Chinese)
[37] CHO A N, JIN Y, AN Y, et al.Microfluidic device with brain extracellular matrix promotes structural and functional maturation of human brain organoids[J].Nature Communications, 2021, 12(1):4730.
[38] NIKOLAEV M, MITROFANOVA O, BROGUIERE N, et al.Homeostatic mini-intestines through scaffold-guided organoid morphogenesi[J].Nature, 2020, 585(7826):574-578.
[39] ACHBERGER K, PROBST C, HADERSPECK J, et al.Merging organoid and organ-on-a-chip technology to generate complex multilayer tissue models in a human retina-on-a-chip platform[J].eLife, 2019, 8:e46188.
[40] TAO T, WANG Y, CHEN W, et al.Engineering humanislet organoids from iPSCs using an organ-on-chip platform[J]. Lab on a Chip, 2019, 19(6):948-958.
[41] QIAN X, JACOB F, SONG M, et al.Generation of human brain region-specific organoids using a miniaturized spinning bioreactor[J].Nature Protocols, 2018, 13(3):565-580.
[42] PHELAN M A, GIANFORCARO A L, GERSTENHABER J A, et al.An air bubble-isolating rotating wall vessel bioreactor for improved spheroid/organoid formation[J].Tissue Engineering Part C-Methods, 2019, 25(8):479-488.
[43] QIAN X, NGUYEN H N, SONG M M, et al.Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure[J].Cell, 2016, 165(5):1238-1254.
[44] DISTEFANO T J, CHEN H Y, PANEBIANCO C, et al.Accelerated and improved differentiation of retinal organoids from pluripotent stem cells in rotating-wall vessel bioreactors[J].Stem Cell Reports, 2021, 16(1):224.
[45] PRZEPIORSKI A, SANDER V, TRAN T, et al.A simple bioreactor-based method to generate kidney organoids frompluripotent stem cells[J].Stem Cell Reports, 2018, 11(2):470-484.
[46] SAITO M, MATSUURA T, MASAKI T, et al.Reconstruction of liver organoid using a bioreact[J].World Journal of Gastroenterology, 2006, 12(12):1881-1888.
[47] LIU H, WANG Y, CUI K, et al.Advances in hydrogels in organoids and organs-on-a-chip[J].Advanced Materials, 2019, 31(50):e1902042.
[48] GIOBBE G G, CROWLEY C, LUNI C, et al.Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture[J].Nature Communications, 2019, 10(1):5658.
[49] MAGNO V, MEINHARDT A, WERNER C.Polymer hydrogels to guide organotypic and organoid cultures[J].Advanced Functional Materials, 2020, 30(48):2000097.
[50] GJOREVSKI N, SACHS A, MANFRIN S, et al.Designer matrices for intestinal stem cell and organoid culture[J]. Nature, 2016, 539(7630):560-564.
[51] YAVITT F M, BROWN T E, HUSHKA E A, et al.The effect of thiol structure on allyl sulfide photodegradable hydrogels and their application as a degradable scaffold for organoid passaging[J].Advanced Materials, 2020, 32(30):e1905366.
[52] CRUZ A R, QUIR M, FARKAS A E, et al.Synthetic hydrogels for human intestinal organoid generation and colonic wound repair[J].Nature Cell Biology, 2017, 19(11):1326-1335.
[53] GIOBBE G G, CROWLEY C, LUNI C, et al.Extracellular matrix hydrogel derived from decellularized tissuesenables endodermal organoid culture[J].Nature Communications, 2019, 10(1):5658.
[54] CAPELING M, CZERWINSKI M, HUANG S, et al.Nonadhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids[J].Stem Cell Reports, 2019, 12(2):381-394.
[55] CANDIELLO J, GRANDHI T S P, GOH S K, et al.3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform[J]. Biomaterials, 2018, 177:27-39.
[56] HE J, ZHANG X, XIA X, et al.Organoid technology for tissue engineering[J].Journal of Molecular Cell Biology, 2020, 12(8):569-579.
[57] MARTI C R, ASHTON R S.The case for applying tissue engineering methodologies to instruct human organoid morphogenesis[J].Acta Biomaterialia, 2017, 54:35-44.
[58] NAKAMURA T, SATO T.Advancing intestinal organoid technology toward regenerative medicine[J].Cellular and Molecular Gastroenterology and Hepatology, 2017, 5(1):51-60.
[59] 赵冰.类器官在器官移植领域的应用前景[J].器官移植, 2022, 13(2):169-175. ZHAO B.Application prospect of organoids in the field of organ transplantation[J].Organ Transplantation, 2022, 13(2):169-175.(in Chinese)
[60] AKKERMAN N, DEFIZE L H.Dawn of the organoid era:3D tissue and organcultures revolutionize the study of development, disease, and regeneration[J]. BioEssays, 2017, 39(4):1600244.
[61] NILSSON H G, MENDES L F, GKLAVA C, et al.Developmentally engineered callus organoid bioassemblies exhibit predictive in vivo long bone healing[J].Advanced Science, 2019, 7(2):1902295.
[62] ROSSEN N S, ANANDAKUMARAN P N, NIEDEN R Z, et al.Injectable therapeutic organoids using sacrificial hydrogels[J].iScience, 2020, 23(5):101052.
[63] LEE J, RABBANI C, GAO H, et al.Hair-bearing human skin generated entirely from pluripotent stem cells[J].Nature, 2020, 582(7812):399-404.
[64] CRUZ A R, QUIR M, FARKAS A E, et al.Synthetic hydrogels for human intestinal organoid generation and colonic wound repair[J].Nature Cell Biology, 2017, 19(11):1326-1335.
[65] JEE J, PARK J H, IM J H, et al.Functional recovery by colon organoid transplantation in a mouse model of radiation proctitis[J]. Biomaterials, 2021, 275, 120925.
[66] TSUCHIDA T, MURATA S, MATSUKI K, et al.The regenerative effect of portal vein injection of liver organoids by retrorsine/partial hepatectomy in rats[J].International Journal of Molecular Sciences, 2019, 21(1):178.
[67] WANG S N, WANG Z, XU T Y, et al.Cerebral organoids repair ischemic stroke brain injury[J].Translation Stroke Research, 2020, 11(5):983-1000.
[68] VONK L A, REEL G, HERNIGOU J, et al.Role of matrix-associated autologous chondrocyte implantation with spheroids in the treatment of large chondral defects in the knee:A systematic review[J].International Journal of Molecular Sciences, 2021, 22(13):7149.
[69] SANTO V E, ESTRADA M F, REBELO S P, et al.Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models[J].Journal of Biotechnology, 2016, 221:118-129.
[70] MURATA D, ARAI K, NAKAYAMA K.Scaffold-free bio-3D printing using spheroids as "bio-inks" for tissue (Re-) construction and drug response tests[J].Advanced Healthcare Materials, 2020, 9(15):e1901831.
[71] LIU H, ZHANG Y, ZHANG Y Y, et al.Human embryonic stem cell-derivedorg-anoid retinoblastoma reveals a cancerous origin[J]. Proceedings of the National Academy of Scinecs of the United States of America, 2020, 117(52):33628-33638.