Mesenchymal stem cell spheroids: potential cell materials for cell therapy

  • Zhongjuan Xu School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, Anhui 230026, China; CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Xingzhi Liu School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, Anhui 230026, China; CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Yu Wei Livingchip Lnc., Nanjing, Jiangsu 211112, China.
  • Zhe Zhao CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Junjun Cao School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, Anhui 230026, China; CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Yong Qiao CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Yanzhen Yu School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, Anhui 230026, China; CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
  • Junjie Zhong Department of Neurosurgery, Fudan University Huashan Hospital, National Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai 200041, China.
  • Guangli Suo CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu 215123, China.
Keywords: Mesenchymal stromal/stem cells, Three-dimensional culture, Spheroids, Stem cell optimization, Regenerative medicine

Abstract

Mesenchymal stromal/stem cells (MSCs) have been applied in clinical trials with an increasing number in recent years. MSCs showed their great potentials in regenerative medicine for their extensive sources, multilineage differentiation potential, low immunogenicity and self-renewal ability. However, the clinical application of MSCs still confronts many challenges including the requirement of large quantity of cells, low survival ability in vivo and the loss of main original characteristics due to two-dimensional (2D) culture although it is beneficial to cells fast expansion. Three-dimensional (3D) culture artificially creates an environment that permits cells to grow or interact with their surroundings in all three dimensions. Therefore, 3D culture was widely regarded as a more preferable and closer physiological microenvironment for cells growth. Recently, many different 3D spheroid culture methods have been developed to optimize MSCs biological characteristics to meet the demand of regenerative medicine. In this review, we comprehensively discussed the merits and demerits of different spheroid formation methods, expounded the mechanisms of spheroid formation and its microenvironment, and illustrated their optimized biological functions and the pre-clinical applications in various tissue injury and regeneration. In the end, we prospected the trends of this research field and proposed the key problems needed to be solved in the future.

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References

Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3(4):393-403.

Wei X, Yang X, Han ZP, Qu FF, Shao L, Shi YF. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol Sin. 2013;34(6):747-54.

Sharma RR, Pollock K, Hubel A, McKenna D. Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion. 2014;54(5):1418-37.

Miceli V, Pampalone M, Vella S, Carreca AP, Amico G, Conaldi PG. Comparison of immunosuppressive and angiogenic properties of human amnion-derived mesenchymal stem cells between 2D and 3D culture systems. Stem Cells Int. 2019;2019:7486279.

Pisciotta A, Bertoni L, Riccio M, Mapelli J, Bigiani A, La Noce M, et al. Use of a 3D floating sphere culture system to maintain the neural crest-related properties of human dental pulp stem cells. Front Physiol. 2018;9:547-47.

Lee EJ, Park SJ, Kang SK, Kim GH, Kang HJ, Lee SW, et al. Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction. Mol Ther. 2012;20(7):1424-33.

Lee M, Song BR, Kim DH, Ha J, Lee M, Choi SJ, et al. Up-regulation of superoxide dismutase 2 in 3D spheroid formation promotes therapeutic potency of human umbilical cord blood-derived mesenchymal stem cells. Antioxidants. 2020;9(1).

Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105(4):1815-22.

Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006;98(5):1076-84.

Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008;8(9):726-36.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7.

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-76.

Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK, Schulman SP, et al. A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol. 2009;54(24):2277-86.

Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 1995;16(4):557-64.

Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med. 1999;5(3):309-13.

Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-28.

Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002;99(13):8932-7.

Togel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol. 2005;289(1):F31-42.

LeBlanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371(9624):1579-86.

Golchin A, Seyedjafari E, Ardeshirylajimi A. Mesenchymal stem cell therapy for COVID-19: present or future. Stem Cell Rev Rep. 2020;16(3):427-33.

Leng Z, Zhu R, Hou W, Feng Y, Yang Y, Han Q, et al. Transplantation of ACE2(-) Mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia. Aging Dis. 2020;11(2):216-28.

Galipeau J. The mesenchymal stromal cells dilemma--does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy. 2013;15(1):2-8.

Regmi S, Pathak S, Kim JO, Yong CS, Jeong JH. Mesenchymal stem cell therapy for the treatment of inflammatory diseases: challenges, opportunities, and future perspectives. Eur J Cell Biol. 2019;98(5-8):151041.

Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009;5(1):54-63.

Wang W, Itaka K, Ohba S, Nishiyama N, Chung UI, Yamasaki Y, et al. 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials. 2009;30(14):2705-15.

Bartosh TJ, Ylostalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K, et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A. 2010;107(31):13724-9.

Frith JE, Thomson B, Genever PG. Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods. 2010;16(4):735-49.

Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med. 2009;206(11):2483-96.

Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 2013;31(2):108-15.

Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J. 2008;3(9-10):1172-84.

Ovsianikov A, Khademhosseini A, Mironov V. The Synergy of Scaffold-Based and Scaffold-Free Tissue Engineering Strategies. Trends Biotechnol. 2018;36(4):348-57.

Sart S, Tsai AC, Li Y, Ma T. Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev. 2014;20(5):365-80.

Xu Y, Shi T, Xu A, Zhang L. 3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney. J Cell Mol Med. 2016;20(7):1203-13.

Peng R, Yao X, Cao B, Tang J, Ding J. The effect of culture conditions on the adipogenic and osteogenic inductions of mesenchymal stem cells on micropatterned surfaces. Biomaterials. 2012;33(26):6008-19.

Tsai AC, Liu Y, Yuan X, Ma T. Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate. Tissue Eng Part A. 2015;21(9-10):1705-19.

Kim J, Ma T. Endogenous extracellular matrices enhance human mesenchymal stem cell aggregate formation and survival. Biotechnol Prog. 2013;29(2):441-51.

Ezquer F, Morales P, Quintanilla ME, Santapau D, Lespay-Rebolledo C, Ezquer M, et al. Intravenous administration of anti-inflammatory mesenchymal stem cell spheroids reduces chronic alcohol intake and abolishes binge-drinking. Sci Rep. 2018;8(1):4325.

Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci. 2015;16(3):5517-27.

Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014;12(4):207-18.

Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev. 2008;14(1):61-86.

Potapova IA, Gaudette GR, Brink PR, Robinson RB, Rosen MR, Cohen IS, et al. Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. Stem Cells. 2007;25(7):1761-8.

Bhang SH, Cho SW, La WG, Lee TJ, Yang HS, Sun AY, et al. Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials. 2011;32(11):2734-47.

Lewis NS, Lewis EE, Mullin M, Wheadon H, Dalby MJ, Berry CC. Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence. J Tissue Eng. 2017;8:2041731417704428.

Cheng NC, Wang S, Young TH. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials. 2012;33(6):1748-58.

Qiao Y, Xu Z, Yu Y, Hou S, Geng J, Xiao T, et al. Single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure. Biomaterials. 2020;227:119573.

Tung YC, Hsiao AY, Allen SG, Torisawa YS, Ho M, Takayama S. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst. 2011;136(3):473-8.

Yang K, Park HJ, Han S, Lee J, Ko E, Kim J, et al. Recapitulation of in vivo-like paracrine signals of human mesenchymal stem cells for functional neuronal differentiation of human neural stem cells in a 3D microfluidic system. Biomaterials. 2015;63:177-88.

Strainiene E, Binkis M, Urnikyte S, Stankevicius V, Sasnauskiene A, Kundrotas G, et al. Microenvironment dependent gene expression signatures in reprogrammed human colon normal and cancer cell lines. BMC cancer. 2018;18(1):222-22.

Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-89.

Kim M, Yun HW, Park DY, Choi BH, Min BH. Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells. Tissue Eng Regen Med. 2018;15(4):427-36.

Tietze S, Krater M, Jacobi A, Taubenberger A, Herbig M, Wehner R, et al. Spheroid culture of mesenchymal stromal cells results in morphorheological properties appropriate for improved microcirculation. Adv Sci. 2019;6(8):1802104.

Baraniak PR, McDevitt TC. Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res. 2012;347(3):701-11.

Zhou Y, Chen H, Li H, Wu Y. 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension. J Cell Mol Med. 2017;21(6):1073-84.

Sun Y, Wang Y, Zhou L, Zou Y, Huang G, Gao G, et al. Spheroid-cultured human umbilical cord-derived mesenchymal stem cells attenuate hepatic ischemia-reperfusion injury in rats. Sci Rep. 2018;8(1):2518.

Dusseiller MR, Schlaepfer D, Koch M, Kroschewski R, Textor M. An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells. Biomaterials. 2005;26(29):5917-25.

Khademhosseini A, Eng G, Yeh J, Fukuda J, Blumling J, 3rd, Langer R, et al. Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment. J Biomed Mater Res A. 2006;79(3):522-32.

Uchida S, Hayakawa K, Ogata T, Tanaka S, Kataoka K, Itaka K. Treatment of spinal cord injury by an advanced cell transplantation technology using brain-derived neurotrophic factor-transfected mesenchymal stem cell spheroids. Biomaterials. 2016;109:1-11.

Huang GS, Dai LG, Yen BL, Hsu SH. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials. 2011;32(29):6929-45.

Wuchter P, Saffrich R, Giselbrecht S, Nies C, Lorig H, Kolb S, et al. Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells. Cell and tissue research. 2016;364(3):573-84.

Chan HF, Zhang Y, Ho YP, Chiu YL, Jung Y, Leong KW. Rapid formation of multicellular spheroids in double-emulsion droplets with controllable microenvironment. Sci Rep. 2013;3:3462.

Halldorsson S, Lucumi E, Gomez-Sjoberg R, Fleming RMT. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens Bioelectron. 2015;63:218-31.

Page H, Flood P, Reynaud EG. Three-dimensional tissue cultures: current trends and beyond. Cell Tissue Res. 2013;352(1):123-31.

Akhmanova M, Osidak E, Domogatsky S, Rodin S, Domogatskaya A. Physical, spatial, and molecular aspects of extracellular matrix of in vivo niches and artificial scaffolds relevant to stem cells research. Stem Cells Int. 2015;2015:167025.

Al-Agele R, Paul E, Taylor S, Watson C, Sturrock C, Drakopoulos M, et al. Physics of animal health: on the mechano-biology of hoof growth and form. J R Soc Interface. 2019;16(155):20190214.

Derda R, Laromaine A, Mammoto A, Tang SK, Mammoto T, Ingber DE, et al. Paper-supported 3D cell culture for tissue-based bioassays. Proc Natl Acad Sci U S A. 2009;106(44):18457-62.

Murphy KC, Hung BP, Browne-Bourne S, Zhou D, Yeung J, Genetos DC, et al. Measurement of oxygen tension within mesenchymal stem cell spheroids. J R Soc Interface. 2017;14(127).

Curcio E, Salerno S, Barbieri G, De Bartolo L, Drioli E, Bader A. Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system. Biomaterials. 2007;28(36):5487-97.

Brophy CM, Luebke-Wheeler JL, Amiot BP, Khan H, Remmel RP, Rinaldo P, et al. Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology. 2009;49(2):578-86.

Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell. 2010;7(2):150-61.

Guo L, Zhou Y, Wang S, Wu Y. Epigenetic changes of mesenchymal stem cells in three-dimensional (3D) spheroids. J Cell Mol Med. 2014;18(10):2009-19.

Li Y, Guo G, Li L, Chen F, Bao J, Shi YJ, et al. Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res. 2015;360(2):297-307.

Imamura A, Kajiya H, Fujisaki S, Maeshiba M, Yanagi T, Kojima H, et al. Three-dimensional spheroids of mesenchymal stem/stromal cells promote osteogenesis by activating stemness and Wnt/beta-catenin. Biochem Biophys Res Commun. 2020;523(2):458-64.

Park MJ, Lee J, Byeon JS, Jeong D-U, Gu N-Y, Cho I-S, et al. Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity. Vet Res Commun. 2018;42(3):171-81.

Murphy KC, Whitehead J, Falahee PC, Zhou D, Simon SI, Leach JK. Multifactorial experimental design to optimize the anti-inflammatory and proangiogenic potential of mesenchymal stem cell spheroids. Stem Cells. 2017;35(6):1493-504.

Huang GS, Hsieh PS, Tseng CS, Hsu SH. The substrate-dependent regeneration capacity of mesenchymal stem cell spheroids derived on various biomaterial surfaces. Biomater Sci. 2014;2(11):1652-60.

Potapova IA, Brink PR, Cohen IS, Doronin SV. Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells. J Biol Chem. 2008;283(19):13100-7.

Dhawan J, Laxman S. Decoding the stem cell quiescence cycle--lessons from yeast for regenerative biology. J Cell Sci. 2015;128(24):4467-74.

Monge C, DiStasio N, Rossi T, Sebastien M, Sakai H, Kalman B, et al. Quiescence of human muscle stem cells is favored by culture on natural biopolymeric films. Stem Cell Res Ther. 2017;8(1):104.

Cheung TH, Rando TA. Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol. 2013;14(6):329-40.

Ben-Sahra I, Manning BD. mTORC1 signaling and the metabolic control of cell growth. Curr Opin Cell Biol. 2017;45:72-82.

Nacarelli T, Sell C. Targeting metabolism in cellular senescence, a role for intervention. Mol Cell Endocrinol. 2017;455(C):83-92.

Carroll B, Nelson G, Rabanal-Ruiz Y, Kucheryavenko O, Dunhill-Turner NA, Chesterman CC, et al. Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing. J Cell Biol. 2017;216(7):1949-57.

Suzuki S, Muneta T, Tsuji K, Ichinose S, Makino H, Umezawa A, et al. Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration. Arthritis Res Ther. 2012;14(3):R136.

Leijten J, Teixeira LSM, Bolander J, Ji W, Vanspauwen B, Lammertyn J, et al. Bioinspired seeding of biomaterials using three dimensional microtissues induces chondrogenic stem cell differentiation and cartilage formation under growth factor free conditions. Scientific reports. 2016;6:36011-11.

Rajaraman G, White J, Tan KS, Ulrich D, Rosamilia A, Werkmeister J, et al. Optimization and scale-up culture of human endometrial multipotent mesenchymal stromal cells: potential for clinical application. Tissue Eng Part C Methods. 2013;19(1):80-92.

Mauck RL, Yuan X, Tuan RS. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage. 2006;14(2):179-89.

Futrega K, Atkinson K, Lott WB, Doran MR. Spheroid coculture of hematopoietic stem/progenitor cells and monolayer expanded mesenchymal stem/stromal cells in polydimethylsiloxane microwells modestly improves in vitro hematopoietic stem/progenitor cell expansion. Tissue Eng Part C Methods. 2017;23(4):200-18.

Schmal O, Seifert J, Schaffer TE, Walter CB, Aicher WK, Klein G. Hematopoietic stem and progenitor cell expansion in contact with mesenchymal stromal cells in a hanging drop model uncovers disadvantages of 3D culture. Stem Cells Int. 2016;2016:4148093.

Huang W, Wang C, Xie L, Wang X, Zhang L, Chen C, et al. Traditional two-dimensional mesenchymal stem cells (MSCs) are better than spheroid MSCs on promoting retinal ganglion cells survival and axon regeneration. Exp Eye Res. 2019;185:107699.

Yan X, Zhang K, Yang Y, Deng D, Lyu C, Xu H, et al. Dispersible and dissolvable porous microcarrier tablets enable efficient large-scale human mesenchymal stem cell expansion. Tissue Eng Part C Methods. 2020;26(5):263-75.

Published
2020-12-13
How to Cite
XuZ., LiuX., WeiY., ZhaoZ., CaoJ., QiaoY., YuY., ZhongJ., & SuoG. (2020). Mesenchymal stem cell spheroids: potential cell materials for cell therapy. STEMedicine, 2(5), e67. https://doi.org/10.37175/stemedicine.v2i5.67
Section
Review articles