Hydrogel-supported poly(L-lactic acid) and polystyrene microsphere-based three-dimensional culture systems for in vitro cell expansion

Abstract

The in vitro expansion of stem cells is important for their application in different life science fields such as cellular tissue and organ repair. An objective of this paper was to achieve static cell culture in vitro through peptide hydrogel-supported microspheres (MSs). The peptides, with their gel-forming properties, microstructures, and mechanical strengths characterized, were found to have good support for the MSs and to be injectable. The internal structures of poly(L-lactic acid) microspheres (PLLA-MSs) and polystyrene microspheres (PS-MSs) made in the laboratory were observed and statistically analyzed in terms of particle size and pore size, following which the co-cultured MSs with cells were found to have good cell adhesion. In addition, three-dimensional (3D) culturing of cells was performed on the peptide and microcarrier composite scaffolds to measure cell viability and cell proliferation. The results showed that the peptides could be stimulated by the culture medium to self-assembly form a 3D fiber network structure. Under the peptide-MS composite scaffold-based cell culture system, further enhancement of the cell culture effect was measured. The peptide-MS composite scaffolds have great potential for the application in 3D cell culture and in vitro cell expansion.

Keywords

microcarrier / microsphere / peptide hydrogel / cell scaffold / three-dimensional culture / cell expansion

References

[1] Zhang Y S, Khademhosseini A . Advances in engineering hydrogels.Science, 2017, 356(6337): eaaf3627

[2] Spicer C D . Hydrogel scaffolds for tissue engineering: the importance of polymer choice.Polymer Chemistry, 2020, 11(2): 184–219

[3] Sun W, Gregory D A, Zhao X . Designed peptide amphiphiles as scaffolds for tissue engineering.Advances in Colloid and Interface Science, 2023, 314: 102866

[4] Curvello R, Kast V, Ordóñez-Morán P, . Biomaterial-based platforms for tumour tissue engineering.Nature Reviews Materials, 2023, 8(5): 314–330

[5] El-Husseiny H M, Mady E A, El-Dakroury W A, . Smart/stimuli-responsive hydrogels: state-of-the-art platforms for bone tissue engineering.Applied Materials Today, 2022, 29: 101560

[6] Zhau H E, Goodwin T J, Chang S M, . Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and PSA expression.In Vitro Cellular & Developmental Biology: Animal, 1997, 33(5): 375–380

[7] Chen F, Le P, Fernandes-Cunha G M, . Bio-orthogonally crosslinked hyaluronate-collagen hydrogel for suture-free corneal defect repair.Biomaterials, 2020, 255: 120176

[8] Wang J, Chu R, Ni N, . The effect of Matrigel as scaffold material for neural stem cell transplantation for treating spinal cord injury.Scientific Reports, 2020, 10(1): 2576

[9] Jin K, Mao X, Xie L, . Transplantation of human neural precursor cells in Matrigel scaffolding improves outcome from focal cerebral ischemia after delayed postischemic treatment in rats.Journal of Cerebral Blood Flow and Metabolism, 2010, 30(3): 534–544

[10] Koutsopoulos S, Zhang S . Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I.Acta Biomaterialia, 2013, 9(2): 5162–5169

[11] Yang Z, Xu H, Zhao X . Designer self-assembling peptide hydrogels to engineer 3D cell microenvironments for cell constructs formation and precise oncology remodeling in ovarian cancer.Advanced Science, 2020, 7(9): 1903718

[12] Li X, Sun Q, Li Q, . Functional hydrogels with tunable structures and properties for tissue engineering applications.Frontiers in Chemistry, 2018, 6: 499

[13] Liu S Q, Tian Q, Hedrick J L, . Biomimetic hydrogels for chondrogenic differentiation of human mesenchymal stem cells to neocartilage.Biomaterials, 2010, 31(28): 7298–7307

[14] Liu S Q, Tian Q, Wang L, . Injectable biodegradable poly(ethylene glycol)/RGD peptide hybrid hydrogels for in vitro chondrogenesis of human mesenchymal stem cells.Macromolecular Rapid Communications, 2010, 31(13): 1148–1154

[15] Liu S Q, Ee P L R, Ke C Y, . Biodegradable poly(ethylene glycol)–peptide hydrogels with well-defined structure and properties for cell delivery.Biomaterials, 2009, 30(8): 1453–1461

[16] Motealleh A, Kehr N S . Nanocomposite hydrogels and their applications in tissue engineering.Advanced Healthcare Materials, 2017, 6(1): 1600938

[17] Elkhoury K, Russell C S, Sanchez-Gonzalez L, . Soft-nanoparticle functionalization of natural hydrogels for tissue engineering applications.Advanced Healthcare Materials, 2019, 8(18): 1900506

[18] Ahmed E M . Hydrogel: preparation, characterization, and applications: a review.Journal of Advanced Research, 2015, 6(2): 105–121

[19] Chen J, Zou X . Self-assemble peptide biomaterials and their biomedical applications.Bioactive Materials, 2019, 4: 120–131

[20] Koutsopoulos S . Self-assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: progress, design guidelines, and applications.Journal of Biomedical Materials Research Part A, 2016, 104(4): 1002–1016

[21] Schop D, van Dijkhuizen-Radersma R, Borgart E, . Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism.Journal of Tissue Engineering and Regenerative Medicine, 2010, 4(2): 131–140

[22] Sart S, Errachid A, Schneider Y J, . Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors.Journal of Tissue Engineering and Regenerative Medicine, 2013, 7(7): 537–551

[23] He Q, Zhang J, Liao Y, . Current advances in microsphere based cell culture and tissue engineering.Biotechnology Advances, 2020, 39: 107459

[24] Zhang L, Zhang J, Ling Y, . Sustained release of melatonin from poly (lactic-co-glycolic acid) (PLGA) microspheres to induce osteogenesis of human mesenchymal stem cells in vitro.Journal of Pineal Research, 2013, 54(1): 24–32

[25] Wang C K, Ho M L, Wang G J, . Controlled-release of rhBMP-2 carriers in the regeneration of osteonecrotic bone.Biomaterials, 2009, 30(25): 4178–4186

[26] Tseng P C, Young T H, Wang T M, . Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension.Biomaterials, 2012, 33(2): 556–564

[27] Qu M, Xiao W, Tian J, . Fabrication of superparamagnetic nanofibrous poly(l-lactic acid)/γ-Fe₂O₃ microspheres for cell carriers.Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2019, 107(3): 511–520

[28] Bai L, Han Q, Han Z, . Stem cells expansion vector via bioadhesive porous microspheres for accelerating articular cartilage regeneration.Advanced Healthcare Materials, 2024, 13(3): 2302327

[29] Lin A, Liu S, Xiao L, . Controllable preparation of bioactive open porous microspheres for tissue engineering.Journal of Materials Chemistry B: Materials for Biology and Medicine, 2022, 10(34): 6464–6471

[30] Maksoud F J, Velázquez de la Paz M F, Hann A J, . Porous biomaterials for tissue engineering: a review.Journal of Materials Chemistry B: Materials for Biology and Medicine, 2022, 10(40): 8111–8165

[31] Chen A K L, Reuveny S, Oh S K W . Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction.Biotechnology Advances, 2013, 31(7): 1032–1046

[32] Kim T K, Yoon J J, Lee D S, . Gas foamed open porous biodegradable polymeric microspheres.Biomaterials, 2006, 27(2): 152–159

[33] Shi X, Jiang J, Sun L, . Hydrolysis and biomineralization of porous PLA microspheres and their influence on cell growth.Colloids and Surfaces B: Biointerfaces, 2011, 85(1): 73–80

[34] Shi X, Sun L, Jiang J, . Biodegradable polymeric microcarriers with controllable porous structure for tissue engineering.Macromolecular Bioscience, 2009, 9(12): 1211–1218

[35] Shi X, Sun L, Gan Z . Formation mechanism of solvent-induced porous PLA microspheres.Acta Polymerica Sinica, 2011, (8): 866–873

[36] Shi X, Cui L, Sun H, . Promoting cell growth on porous PLA microspheres through simple degradation methods.Polymer Degradation & Stability, 2019, 161: 319–325

[37] Chiu Y C, Cheng M H, Engel H, . The role of pore size on vascularization and tissue remodeling in PEG hydrogels.Biomaterials, 2011, 32(26): 6045–6051

[38] Huang C C, Wei H J, Yeh Y C, . Injectable PLGA porous beads cellularized by hAFSCs for cellular cardiomyoplasty.Biomaterials, 2012, 33(16): 4069–4077

[39] Andersson A S, Bäckhed F, von Euler A, . Nanoscale features influence epithelial cell morphology and cytokine production.Biomaterials, 2003, 24(20): 3427–3436

[40] Loesberg W A, te Riet J, van Delft F C, . The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.Biomaterials, 2007, 28(27): 3944–3951

[41] Rangarajan S, Madden L, Bursac N . Use of flow, electrical, and mechanical stimulation to promote engineering of striated muscles.Annals of Biomedical Engineering, 2014, 42(7): 1391–1405

[42] Berenzi A, Steimberg N, Boniotti J, . MRT letter: 3D culture of isolated cells: a fast and efficient method for optimizing their histochemical and immunocytochemical analyses.Microscopy Research and Technique, 2015, 78(4): 249–254

[43] Bueno E M, Bilgen B, Barabino G A . Wavy-walled bioreactor supports increased cell proliferation and matrix deposition in engineered cartilage constructs.Tissue Engineering, 2005, 11(11–12): 1699–1709

Declaration of competing interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (Grant No. 2021YFC2101400).

RIGHTS & PERMISSIONS

2024 Higher Education Press