INVESTIGATION OF 3D CULTURE OF HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS IN A MICROFLUIDIC PLATFORM

Year 2021, Volume: 22 Issue: Vol:22- 8th ULPAS - Special Issue 2021, 85 - 97, 30.11.2021

Ceren Özel Yücel Koç Ahmet Topal , Aliakbar Ebrahimi , Tayfun Şengel , Hamed Ghorbanpoor , Fatma Doğan Guzel , Onur Uysal , Ayla Eker Sarıboyacı , Hüseyin Avcı

https://doi.org/10.18038/estubtda.983881

Cited By: 2

Abstract

Mesenchymal stem cells (MSCs) are multipotent stem cells that can support various tissues including bone marrow, adipose tissue, and synovial fluids, from which they can be readily isolated. The objective of this study is to harness the advantages of microfluidic systems for controlling and enhancing the maintenance and viability, and regenerative properties of MSCs by providing a 3D culture microenvironment with gelatin methacrylate (GelMA) hydrogel and exposing the cells to a slow fluid flow and low shear stress conditions. GelMA has methacryloyl groups and can be crosslinked by a photocuring process using biocompatible photoinitiators. The most common used photoinitiator for cellular encapsulation within hydrogels is the ultraviolet (UV) initiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959 or I2959), but due to its low water solubility and the necessity of using a shorter wavelength light (365 nm), it can lead to cellular phototoxic and genotoxic effects. To overcome these limitations, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) have recently been used with GelMA as an alternative photoinitiator. Because LAP is highly water soluble and has a 10 times faster polymerization rate, and it requires a visible light (λ = 405 nm) which makes it much safer for the cells, we use 10% GelMA together with 0.05% LAP photoinitiator for bioprinting human adipose tissue derived MSCs (hAT-MSCs) onto a membrane that has a 40 µm mesh size. To demonstrate a microfluidic culture advancement for improving the biological activities and regenerative capacity of the cells including cell adhesion, growth, viability and proliferation capacity as ultimate goals of this study, the membrane carrying the bioprinted construct was placed in a PDMS microchannel and exposed to the fluid to obtain dynamic microenvironments found in the human body. As a result, the cells were successfully maintained in the microfluidic 3D cell culture for two days, with a high cell viability of 99%.

Keywords

MSCs, GelMA, 3D cell culture, cell adhesion, Organ on a chip

Supporting Institution

TÜBİTAK

Project Number

20AG003 and 20AG031

Thanks

This study was supported by Turkish Scientific and Technological Council (TÜBİTAK 1004-Regenerative and Restorative Medicine Research and Applications) under the grant numbers of 20AG003 and 20AG031.

INVESTIGATION OF 3D CULTURE OF HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS IN A MICROFLUIDIC PLATFORM

Abstract

Project Number

20AG003 and 20AG031

References

• [1] Ingber DE. Developmentally Inspired Human ‘Organs on Chips’. Development, 2018; 145(16):dev156125.
• [2] Bhatia SN, Ingber DE. Microfluidic Organs-on-Chips. Nat Biotechnol, 2014; 32(8):760-72.
• [3] Avci H, Güzel FD, Erol S, Akpek A. Recent Advances in Organ-on-A-Chip Technologies and future Challenges: A Review. Turkish Journal of Chemistry, 2017; 42(3):587-610.
• [4] Zhang YS, Aleman J, Shin SR. et al. Multisensor-Integrated Organs-on-Chips Platform for Automated and Continual in Situ Monitoring of Organoid Behaviors. Proceedings of the National Academy of Sciences, 2017; 114(12):E2293-E2302.
• [5] Shin SR, Zhang YS, Kim D-J. et al. Aptamer-Based Microfluidic Electrochemical Biosensor for monitoring Cell-Secreted Trace Cardiac Biomarkers. Analytical Chemistry, 2016; 88(20):10019-10027.
• [6] Shin SR, Kilic T, Zhang YS. et al. Label‐Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes. Advanced Science, 2017; 4(5):1600522.
• [7] Mancio-Silva L, Fleming HE, Miller AB. et al. Improving Drug Discovery by Nucleic Acid Delivery in Engineered Human Microlivers. Cell Metabolism, 2019; 29(3):727-735. e3.
• [8] de Souza N. Organoids. Nature Methods, 2018; 15(1):23-23.
• [9] Garbioglu DB, Demir N, Ozel C, Avci H, Dincer M. Determination of Therapeutic Agents Efficiencies of Microsatellite Instability High Colon Cancer Cells in Post‐Metastatic Liver Biochip Modeling. The FASEB Journal, 2021; 35(9):e21834.
• [10] Uccelli A, Moretta L, Pistoia V. Mesenchymal Stem Cells in Health and Disease. Nature Reviews Immunology, 2008; 8(9):726-736.
• [11] Pers YM, Ruiz M, Noël D, Jorgensen C. Mesenchymal Stem Cells for the Management of Inflammation in Osteoarthritis: State of The Art and Perspectives. Osteoarthritis and Cartilage, 2015; 23(11):2027-2035.
• [12] Topal AE. Mesenchymal Stem Cell Mechanics on Osteoinductive Peptide Nanofibers. PhD, Bilkent University, Ankara, Turkey, 2017.
• [13] Topal AE, Tansik G, Ozkan AD, Guler MO, Dana A, Tekinay AB. Nanomechanical Characterization of Osteogenic Differentiation of Mesenchymal Stem Cells on Bioactive Peptide Nanofiber Hydrogels. Advanced Materials Interfaces, 2017; 4(20):1700090.
• [14] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell, 2006; 126(4):677-689.
• [15] Mousavi SJ, Hamdy Doweidar M. Role of Mechanical Cues in cell Differentiation and Proliferation: a 3D Numerical Model. PloS one, 2015; 10(5):e0124529.
• [16] Zhao F, Chella R, Ma T. Effects of Shear Stress on 3‐D human Mesenchymal Stem Cell Construct Development in a Perfusion Bioreactor System: Experiments and Hydrodynamic Modeling. Biotechnology and Bioengineering, 2007; 96(3):584-595.
• [17] Higuera GA, van Boxtel A, van Blitterswijk CA, Moroni L. The Physics of Tissue Formation with Mesenchymal Stem Cells. Trends in Biotechnology, 2012; 30(11):583-590. [18] Riddle RC, Taylor AF, Genetos DC, Donahue HJ. MAP Kinase and Calcium Signaling Mediate Fluid Flow-Induced Human Mesenchymal Stem Cell Proliferation. American Journal of Physiology-Cell Physiology, 2006; 290(3):C776-C784.
• [19] Dash SK, Sharma V, Verma RS, Das SK. Low Intermittent Flow Promotes Rat Mesenchymal Stem Cell Differentiation in Logarithmic Fluid Shear Device. Biomicrofluidics 2020; 14(5):054107. [20] Kim KM, Choi YJ, Hwang J-H. et al. Shear Stress Induced by An Interstitial Level of Slow Flow Increases the Osteogenic Differentiation of Mesenchymal Stem Cells Through TAZ Activation. PloS one, 2014; 9(3):e92427.
• [21] Bissoyi A, Bit A, Singh BK, Singh AK, Patra PK. Enhanced Cryopreservation of MSCs in Microfluidic Bioreactor by Regulated Shear Flow. Scientific Reports, 2016; 6(1):1-13.
• [22] Zhang J, Wei X, Zeng R, Xu F, Li X. Stem Cell Culture and Differentiation in Microfluidic Devices Toward Organ-on-A-Chip. Future Science OA, 2017; 3(2):FSO187.
• [23] Costa‐Almeida R, Domingues RM, Fallahi A. et al. Cell‐Laden Composite Suture Threads for Repairing Damaged Tendons. Journal of Tissue Engineering and Regenerative Medicine, 2018; 12(4):1039-1048.
• [24] Lim KS, Galarraga JH, Cui X, Lindberg GC, Burdick JA, Woodfield TB. Fundamentals and Applications of Photo-Cross-Linking In Bioprinting. Chemical Reviews, 2020; 120(19):10662-10694.
• [25] Wong DY, Ranganath T, Kasko AM. Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells. PloS one, 2015; 10(9):e0139307.
• [26] Lawrence KP, Douki T, Sarkany RP, Acker S, Herzog B, Young AR. The UV/Visible Radiation Boundary Region (385–405 nm) Damages Skin Cells and Induces “dark” Cyclobutane Pyrimidine Dimers in Human Skin in vivo. Scientific Reports, 2018; 8(1):1-12.
• [27] Rajabi N, Rezaei A, Kharaziha M. et al. Recent advances on bioprinted gelatin methacrylate-based hydrogels for tissue repair. Tissue Engineering Part A. 2021; 27(11-12):679-702.
• [28] Yin J, Yan M, Wang Y, Fu J, Suo H. 3D Bioprinting of low-Concentration Cell-Laden Gelatin Methacrylate (GelMA) Bioinks with A Two-Step Cross-Linking Strategy. ACS Applied Materials & Interfaces, 2018; 10(8):6849-6857.
• [29] Miri AK, Nieto D, Iglesias L. et al. Microfluidics‐Enabled Multimaterial Maskless Stereolithographic Bioprinting. Advanced Materials, 2018; 30(27):1800242.
• [30] Didarian R, Ebrahimi A, Ghorbanpoor H, Dizaji AN, Hashempour H, Guzel FD, Avci H. Investigation of Polar and Nonpolar Cyclotides Separation from Violet Extract Through Microfluidic Chip. 8. International Fiber and Polymer Research Symposium; 18-19 June 2021, Eskişehir Osmangazi University, Eskisehir, Turkey.
• [31] Gupta NS, Lee K-S, Labouriau AJP. Tuning Thermal and Mechanical Properties of Polydimethylsiloxane with Carbon Fibers. Polymers, 2021; 13(7):1141.
• [32] Dizaji AN, Ozturk Y, Ghorbanpoor H. et al. Investigation of the Effect of Channel Structure and Flow Rate on On-Chip Bacterial Lysis. IEEE Trans Nanobioscience, 2021; 20(1):86-91.
• [33] Kaur J, Ghorbanpoor H, Öztürk Y. et al. On‐Chip Label‐Free Impedance‐Based Detection of Antibiotic Permeation. IET Nanobiotechnology, 2021; 15(1):100-106.
• [34] Ozdemir AT, Ozgul Ozdemir RB, Kirmaz C. et al. The Paracrine Immunomodulatory Interactions Between the Human Dental Pulp Derived Mesenchymal Stem Cells and CD4 T Cell Subsets. Cell Immunol, 2016; 310:108-115.
• [35] Gao G, Schilling AF, Hubbell K. et al. Improved Properties of Bone and Cartilage Tissue from 3D Inkjet-Bioprinted Human Mesenchymal Stem Cells by Simultaneous Deposition and Photocrosslinking in PEG-GelMA. Biotechnology Letters, 2015; 37(11):2349-2355.
• [36] Dupuy A, Ju LA, Passam FH. Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow. Bio-protocol, 2019; 9(6):e3195.
• [37] Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z. Stem Cells: Past, Present, and Future. Stem Cell Research & Therapy, 2019; 10(1):1-22.
• [38] Park D, Lim J, Park JY, Lee S-H. Concise Review: Stem Cell Microenvironment on A Chip: Current Technologies for Tissue Engineering and Stem Cell Biology. Stem Cells Translational Medicine, 2015; 4(11):1352-1368.
• [39] Dominici M, Le Blanc K, Mueller I. et al. Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society for Cellular Therapy Position Statement Cytotherapy, 2006; 8(4):315-7.
• [40] Karaöz E, Demircan PÇ, Erman G, Güngörürler E, Sarıboyacı AE. Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells. Turkish Journal of Hematology, 2017; 34(3):213. [41] Özdemir RBÖ, Özdemir AT, Sarıboyacı AE, Uysal O, Tuğlu Mİ, Kırmaz C. The Investigation of Immunomodulatory Effects of Adipose Tissue Mesenchymal Stem Cell Educated Macrophages on The CD4 T Cells. Immunobiology, 2019; 224(4):585-594.
• [42] Wade RJ, Burdick JA. Engineering ECM Signals into Biomaterials. Materials Today, 2012; 15(10):454-459.
• [43] Choi CK, Breckenridge MT, Chen CS. Engineered Materials and The Cellular Microenvironment: A Strengthening Interface Between Cell Biology and Bioengineering. Trends in Cell Biology, 2010; 20(12):705-714.
• [44] Xiao S, Zhao T, Wang J et al. Gelatin methacrylate (GelMA)-Based Hydrogels for Cell Transplantation: An Effective Strategy for Tissue Engineering. Stem Cell Reviews and Reports, 2019; 15(5):664-679.
• [45] Winer JP, Janmey PA, McCormick ME, Funaki M. Bone Marrow-Derived Human Mesenchymal Stem Cells Become Quiescent on Soft Substrates But Remain Responsive to Chemical or Mechanical Stimuli. Tissue Engineering Part A, 2009; 15(1):147-154.
• [46] Dhall A, Lab C. Simulating Fluid Flow Through a Culture Chip for Cell Migration Studies in Microgravity. COMSOL Conference; 2016, Boston, USA.
• [47] Song MJ, Brady-Kalnay SM, McBride SH, Phillips-Mason P, Dean D, Knothe Tate ML. Mapping The Mechanome of Live Stem Cells using A Novel Method to Measure Local Strain Fields in Situ at the Fluid-Cell Interface. PLoS One, 2012; 7(9):e43601.
• [48] Song MJ, Dean D, Knothe Tate ML. In Situ Spatiotemporal Mapping of Flow Fields Around Seeded Stem Cells at The Subcellular Length Scale. PLoS one, 2010; 5(9):e12796.
• [49] McBride SH, Falls T, Knothe Tate ML. Modulation of stem cell shape and fate B: Mechanical Modulation of Cell Shape and Gene Expression. Tissue Engineering Part A, 2008; 14(9):1573-1580.
• [50] Tate MLK, Falls TD, McBride SH, Atit R, Knothe UR. Mechanical Modulation of Osteochondroprogenitor Cell Fate. The International Journal of Biochemistry & Cell Biology, 2008; 40(12):2720-2738.