Three-dimensional (3D) cell culture is gaining attention for replicating in vivo conditions better than traditional two-dimensional (2D) cultures. The hanging drop method is a simple 3D culture technique with significant potential due to its ease of use. This study compares mesenchymal stem cells’ morphology, viability, surface markers and pluripotency gene of Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) from the umbilical cord in 2D and 3D cultures.
Methods
Umbilical cord MSCs were cultured using the hanging drop technique for 3D culture and 2D culture. Morphology and viability were observed while surface marker analysis was performed using flow cytometry. The harvested cells were then extracted and underwent RNA isolation, cDNA synthesis, and examination of the expression of the OCT4 and SOX2.
Results
Significant differences between 2D and 3D cultures regarding morphology, viability, and CD expression were observed. Spheroids in 3D culture exhibited a significant increase in diameter over time (p<0.0001), while CD73 expression was higher in 2D cultures compared to 3D (p = 0.0225). In 2D culture cells, the OCT4 gene was expressed more than in 3D culture (p = 0.0286).
Conclusions
These findings suggest that 3D culture provides valuable insights into stem cell behavior, with important implications for regenerative medicine.
HoangDMPhamPTBachTQ, et al.Stem cell-based therapy for human diseases. Signal Transduct Target Ther2022; 7: 1–41.
2.
WangJDengGWangS, et al.Enhancing regenerative medicine: the crucial role of stem cell therapy. Front Neurosci2024; 18: 1269577.
3.
ZakrzewskiWDobrzyńskiMSzymonowiczM, et al.Stem cells: past, present, and future. Stem Cell Res Ther2019; 10: 68.
4.
BuenoCMartínez-MorgaMGarcía-BernalD, et al.Differentiation of human adult-derived stem cells towards a neural lineage involves a dedifferentiation event prior to differentiation to neural phenotypes. Sci Rep2021; 11: 12034.
5.
NoviantariARinendyaputriRAriyantoI. Differentiation ability of rat-mesenchymal stem cells from bone marrow and adipose tissue to neurons and glial cells. Indones J Biotechnol2020; 25: 43–51.
6.
HernándezRJiménez-LunaCPerales-AdánJ, et al.Differentiation of human mesenchymal stem cells towards neuronal lineage: clinical trials in nervous system disorders. Biomol Ther (Seoul)2020; 28: 34–44.
7.
AhmedSSethNGoyalR, et al.Induced pluripotent stem cells: a game-changer in dentistry’s regenerative landscape. J Cell Biotechnol2024; 10: 131–137.
8.
ShangYGuanHZhouF. Biological characteristics of umbilical cord mesenchymal stem cells and its therapeutic potential for hematological disorders. Front Cell Dev Biol2021; 9: 570179.
9.
YeaJHKimYJoCH. Comparison of mesenchymal stem cells from bone marrow, umbilical cord blood, and umbilical cord tissue in regeneration of a full-thickness tendon defect in vitro and in vivo. Biochem Biophys Rep2023; 34: 101486.
10.
ChangNC. Autophagy and stem cells: self-eating for self-renewal. Front Cell Dev Biol2020; 8: 138.
11.
SachdevaSSalujaH. Mesenchymal cells as the biological modifiers for stimulation and regulation of cellular and molecular healing and regeneration. J Cell Biotechnol2024; 10: 109–124.
12.
FuXXuBJiangJ, et al.Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells. Clin Proteomics2020; 17: 15.
13.
ShenCYangCXuS, et al.Comparison of osteogenic differentiation capacity in mesenchymal stem cells derived from human amniotic membrane (AM), umbilical cord (UC), chorionic membrane (CM), and decidua (DC). Cell Biosci2019; 9: 17.
14.
GhaneialvarHSoltaniLRahmaniHR, et al.Characterization and classification of mesenchymal stem cells in several species using surface markers for cell therapy purposes. Indian J Clin Biochem2018; 33: 46–52.
15.
SandraFSudionoJFeterY, et al.Investigation on cell surface markers of dental pulp stem cell isolated from impacted third molar based on International Society for Cellular Therapy proposed mesenchymal stem cell markers. Mol Cell Biomed Sci2019; 3: 1–6.
16.
MebarkiMAbadieCLargheroJ, et al.Human umbilical cord-derived mesenchymal stem/stromal cells: a promising candidate for the development of advanced therapy medicinal products. Stem Cell Res Ther2021; 12: 152.
17.
BaghaeiKHashemiSMTokhanbigliS, et al.Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow. Gastroenterol Hepatol Bed Bench2017; 10: 208–213.
18.
EggerDOliveiraACMallingerB, et al.From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix. Stem Cell Res Ther2019; 10: 1–10.
19.
KyriakopoulouKKoutsakisCPiperigkouZ, et al.Recreating the extracellular matrix: novel 3D cell culture platforms in cancer research. FEBS J2023; 290: 5238–5247.
20.
JensenCTengY. Is it time to start transitioning from 2D to 3D cell culture?Front Mol Biosci2020; 7: 33.
21.
KapałczyńskaMKolendaTPrzybyłaW, et al.2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci2018; 14: 910–919.
22.
SunMLiuAYangX, et al.3D cell culture—can it Be as popular as 2D cell culture?Advanced NanoBiomed Research2021; 1: 2000066.
23.
CacciamaliAVillaRDottiS. 3D cell cultures: evolution of an ancient tool for new applications. Front Physiol2022; 13: 836480.
24.
BijuTSPriyaVVFrancisAP. Role of three-dimensional cell culture in therapeutics and diagnostics: an updated review. Drug Deliv Transl Res2023; 13: 2239–2253.
25.
AbuwatfaWHPittWGHusseiniGA. Scaffold-based 3D cell culture models in cancer research. J Biomed Sci2024; 31: 7.
26.
ParkYHuhKMKangSW. Applications of biomaterials in 3D cell culture and contributions of 3D cell culture to drug development and basic biomedical research. Int J Mol Sci2021; 22: 2491.
27.
WangHBrownPCChowECY, et al.3D cell culture models: drug pharmacokinetics, safety assessment, and regulatory consideration. Clin Transl Sci2021; 14: 1659–1680.
28.
HabanjarODiab-AssafMCaldefie-ChezetF, et al.3D cell culture systems: tumor application, advantages, and disadvantages. Int J Mol Sci2021; 22: 12200.
ChoC-YChiangT-HHsiehL-H, et al.Development of a novel hanging drop platform for engineering controllable 3D microenvironments. Front Cell Dev Biol2020; 8: 327.
31.
HuangBChengXWangH, et al.Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively. J Transl Med2016; 14: 1–12.
32.
DingYYuanXZouY, et al.OCT4, SOX2 and NANOG co-regulate glycolysis and participate in somatic induced reprogramming. Cytotechnology2022; 74: 371–383.
33.
SwainNThakurMPathakJ, et al.SOX2, OCT4 and NANOG: the core embryonic stem cell pluripotency regulators in oral carcinogenesis. J Oral Maxillofac Pathol2020; 24: 368–373.
34.
SibueaCVHutabaratESKSimangunsongDM. Viabilitas hepatosit pada monokultur 3D metode hanging drop dan monokultur 2D. Nommensen Journal of Medicine2022; 7: 36–38.
35.
ZhangMGuLZhengP, et al.Improvement of cell counting method for Neubauer counting chamber. J Clin Lab Anal2020; 34: e23024.
36.
RenesmeLPierroMCobeyKD, et al.Definition and characteristics of mesenchymal stromal cells in preclinical and clinical studies: a scoping review. Stem Cells Transl Med2022; 11: 44–54.
37.
ChaicharoenaudomrungNKunhormPNoisaP. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells2019; 11: 1065–1083.
38.
KorzyńskaAZychowiczM. A method of estimation of the cell doubling time on basis of the cell culture monitoring data. Biocybern Biomed Eng2008; 28: 75–82.
39.
HabanjarODiab-AssafMCaldefie-ChezetF, et al.3D cell culture systems: tumor application, advantages, and disadvantages. Int J Mol Sci2021; 22: 12200.
40.
ShichiYSasakiNMichishitaM, et al.Enhanced morphological and functional differences of pancreatic cancer with epithelial or mesenchymal characteristics in 3D culture. Sci Rep2019; 9: 10871.
41.
AntoniDBurckelHJossetE, et al.Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci2015; 16: 5517–5527.
42.
BiałkowskaKKomorowskiPBryszewskaM, et al.Spheroids as a type of three-dimensional cell cultures—examples of methods of preparation and the most important application. Int J Mol Sci2020; 21: 6225.
Juarez-MorenoKChávez-GarcíaDHirataG, et al.Monolayer (2D) or spheroids (3D) cell cultures for nanotoxicological studies? Comparison of cytotoxicity and cell internalization of nanoparticles. Toxicol Vitro2022; 85: 105461.
45.
PalmWThompsonCB. Nutrient acquisition strategies of mammalian cells. Nature2017; 546: 234–242.
46.
RademakersTHorvathJMvan BlitterswijkCA, et al.Oxygen and nutrient delivery in tissue engineering: approaches to graft vascularization. J Tissue Eng Regen Med2019; 13: 1815–1829.
47.
BiałkowskaKKomorowskiPBryszewskaM, et al.Spheroids as a type of three-dimensional cell cultures-examples of methods of preparation and the most important application. Int J Mol Sci2020; 21: 6225.
48.
ZingalesVEspositoMRQuagliataM, et al.Comparative study of spheroids (3D) and monolayer cultures (2D) for the in vitro assessment of cytotoxicity induced by the mycotoxins sterigmatocystin, ochratoxin A and patulin. Foods2024; 13: 564.
49.
SonYBBhartiDKimSB, et al.Comparison of pluripotency, differentiation, and mitochondrial metabolism capacity in three-dimensional spheroid formation of dental pulp-derived mesenchymal stem cells. BioMed Res Int2021; 2021: 5540877.
50.
UrzìOGasparroRCostanzoE, et al.Three-dimensional cell cultures: the bridge between in vitro and in vivo models. Int J Mol Sci2023; 24: 12046.
51.
SoaresCPMidlejVOliveiraMEW, et al.2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression. PLoS One2012; 7: e38147.
HuangSWTzengSCChenJK, et al.A dynamic hanging-drop system for mesenchymal stem cell culture. Int J Mol Sci2020; 21: 4298.
54.
DuvalKGroverHHanLH, et al.Modeling physiological events in 2D vs. 3D cell culture. Physiology2017; 32: 266–277.
55.
AlmelaTTayebiLMoharamzadehK. 3D bioprinting for in vitro models of oral cancer: toward development and validation. Bioprinting2021; 22: e00132.
56.
TidwellTRRøslandGVTronstadKJ, et al.Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines. Cancer Metab2022; 10: 9.
57.
JensenCTengY. Is it time to start transitioning from 2D to 3D cell culture?Front Mol Biosci2020; 7: 33.
58.
AbbasZNAl-SaffarAZJasimSM, et al.Comparative analysis between 2D and 3D colorectal cancer culture models for insights into cellular morphological and transcriptomic variations. Sci Rep2023; 13: 18380.
59.
ZhangSLiJLiC, et al.CD73-Positive small extracellular vesicles derived from umbilical cord mesenchymal stem cells promote the proliferation and migration of pediatric urethral smooth muscle cells through adenosine pathway. Front Bioeng Biotechnol2022; 10: 895998.
60.
TanKZhuHZhangJ, et al.CD73 expression on mesenchymal stem cells dictates the reparative properties via its anti-inflammatory activity. Stem Cells Int2019; 2019: 8717694.
61.
RybkowskaPRadoszkiewiczKKawalecM, et al.The metabolic changes between monolayer (2D) and three-dimensional (3D) culture conditions in human mesenchymal stem/stromal cells derived from adipose tissue. Cells2023; 12: 178.
62.
PetrukNTuominenSÅkerfeltM, et al.CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer. Sci Rep2021; 11: 6035.
63.
BachNWinzerRTolosaE, et al.The clinical significance of CD73 in cancer. Int J Mol Sci2023; 24: 11759.
64.
SauzayCVoutetakisKChatziioannouA, et al.CD90/Thy-1, a cancer-associated cell surface signaling molecule. Front Cell Dev Biol2019; 7: 66.
65.
ZhangKCheSSuZ, et al.CD90 promotes cell migration, viability and sphere-forming ability of hepatocellular carcinoma cells. Int J Mol Med2018; 41: 946–954.
66.
HigaRHanadaTTeranishiH, et al.CD105 maintains the thermogenic program of beige adipocytes by regulating Smad2 signaling. Mol Cell Endocrinol2018; 474: 184–193.
67.
BaeY-JKwonY-RKimHJ, et al.Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine. Blood Res2017; 52: 18–24.
68.
LouYGuoDZhangH, et al.Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression. Open Life Sci2016; 11: 519–523.
69.
ThakurGBokE-YKimS-B, et al.Scaffold-free 3D culturing enhance pluripotency, immunomodulatory factors, and differentiation potential of Wharton’s jelly-mesenchymal stem cells. Eur J Cell Biol2022; 101: 151245.
70.
MaticIAntunovicMBrkicS, et al.Expression of OCT-4 and SOX-2 in bone marrow-derived human mesenchymal stem cells during osteogenic differentiation. Open Access Maced J Med Sci2016; 4: 9–16.
