Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters

Decarli, Monize Caiado; Castro, Mateus Vidigal de; Nogueira, Júlia A.; Nagahara, Mariana Harue Taniguchi; Westin, Cecília Buzatto; Oliveira, Alexandre Leite Rodrigues de; Silva, Jorge Vicente L.; Moroni, Lorenzo; Mota, Carlos; Moraes, Ângela Maria

doi:10.1016/j.msec.2022.112685

Cited by 11 publications

(8 citation statements)

References 55 publications

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“…The microwell array insert used to produce hMSC spheroids was previously described in detail. [63] Briefly, using a cast previously obtained by additive manufacturing, several array inserts were obtained through mi-cromolding, using a non-adhesive hydrogel made of sterile ultrapure 3% (w/v) agarose (16 500 100, Invitrogen) in PBS solution. The microwell array inserts were manufactured the day before cell seeding, immersed in basic culture medium and maintained in the incubator overnight.…”

Section: Scaffold Shape Stability Analysis In Culture Mediummentioning

confidence: 99%

Bioprinting of Stem Cell Spheroids Followed by Post‐Printing Chondrogenic Differentiation for Cartilage Tissue Engineering

Decarli

Seijas‐Gamardo

Morgan

et al. 2023

Adv Healthcare Materials

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Cartilage tissue presents low self‐repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post‐bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum‐alginate hydrogel. Multi‐layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof‐of‐concept of their potential use as stable chondral tissue implants.

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Section: Scaffold Shape Stability Analysis In Culture Mediummentioning

confidence: 99%

Bioprinting of Stem Cell Spheroids Followed by Post‐Printing Chondrogenic Differentiation for Cartilage Tissue Engineering

Decarli

Seijas‐Gamardo

Morgan

et al. 2023

Adv Healthcare Materials

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Section: Introductionmentioning

confidence: 99%

“…To scale up and carry out medication cytotoxicity and efficacy testing as well as tissue engineering techniques, three-dimensional cellular aggregates must be created through controlled and reproducible processes [ 13 ]. The microenvironment of tissues and organs in nature can be mimicked by these aggregates [ 13 ]. Spheroids of bone marrow stromal cells and dental pulp stem cells, with good cell viability and average diameters of around 253 and 220 μm, respectively, were made using the equipment with the form that was thought to be most appropriate [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…The microenvironment of tissues and organs in nature can be mimicked by these aggregates [ 13 ]. Spheroids of bone marrow stromal cells and dental pulp stem cells, with good cell viability and average diameters of around 253 and 220 μm, respectively, were made using the equipment with the form that was thought to be most appropriate [ 13 ]. The primary objective of this study was to evaluate the effects of an enamel matrix derivative on the viability, osteogenic differentiation, and mineralization of human mesenchymal stem cell–derived cell spheroids.…”

Section: Introductionmentioning

confidence: 99%

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Effects of Enamel Matrix Derivative on Cell Spheroids Made of Stem Cells Obtained from the Gingiva on Osteogenic Differentiation

Hwa

Lee

et al. 2023

Medicina

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Background and Objectives: A derivative of the enamel matrix was used to speed up periodontal regeneration, including the formation of new cementum, alveolar bone, and periodontal ligament. In this study, human gingiva-derived stem cell–derived cell spheroids were used to assess the effects of an enamel matrix derivative on cell viability, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then coupled with unloaded control groups and an enamel matrix derivative at a final concentration of 2.7, 27, 270, and 2700 μg/mL. The morphological examination of the created stem cell spheroids took place on days 1, 3, 5, and 7. The Live/Dead Kit assay was used to determine the qualitative viability of cells on days 3 and 7. Using the Cell Counting Kit-8, the quantitative vitality of the cell spheroids was assessed on days 1, 3, and 5. On days 7 and 14, alkaline phosphatase activity assays and Alizarin Red S staining were carried out to examine the osteogenic differentiation of the cell spheroids. RUNX2 and COL1A1 expression levels on days 7 and 14 were determined using real-time polymerase chain reaction. Results: The added enamel matrix derivative at the tested concentrations did not significantly alter the morphology of the applied stem cells’ well-formed spheroids on day 1. On days 3 and 7, the majority of the spheroids’ cells fluoresced green while they were being cultivated. Alkaline phosphatase activity data revealed a substantial rise in the 2700 μg/mL group on day 7 when compared to the unloaded control (p < 0.05). On days 7 and 14, calcium deposits were distinctly seen in each group. In the 27 and 2700 μg/mL groups, the treatment with the enamel matrix derivative resulted in noticeably higher values for the Alizarin Red S staining (p < 0.05). qPCR results showed that adding an enamel matrix derivative to the culture of the 27 μg/mL group raised the level of RUNX2 mRNA expression. Conclusions: These results lead us to the conclusion that a derivative of the enamel matrix may be used to promote osteogenic differentiation in stem cell spheroids.

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“…In several drug delivery research papers, 2D and 3D cultures were compared and presented; thus, the differences and effects in 2D were insignificant, but these difference in results in 3D were noticeable and proved to be useful materials for many applications [ 6 , 7 ]. Three-dimensional (3D) cell aggregates can imitate the natural microenvironment [ 8 ]. Small adipose-derived stem cell spheroids cultured in scaffold-free three-dimensional condition survived and promoted bone regeneration under in vitro and in vivo conditions [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Assessment of the Impacts of Centipeda minima (L.) on Cell Viability, and Osteogenic Differentiation of Mesenchymal Stem Cell Spheroids

Lee

Uddin³

et al. 2022

Medicina

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Background and Objectives: Centipeda minima (L.) is a well-known and traditional pharmaceutical that has been utilized to treat different conditions controlling rhinitis, soothe pain, and decrease swelling. We assessed the impacts of Centipeda minima (L.) extricates (CMTs) on the osteogenic differentiation of cell spheroids made of human-bone-marrow-derived mesenchymal stem cells. Materials and Methods: Mesenchymal stem cells (MSCs) in spheroid 3D culture were generated and propagated in the presence of CMTs ranging from 0 to 1 μg/mL. Cell morphology was measured on Days 1, 3, 5, and 7. The quantitative cellular viability was evaluated on Days 1, 3, 5, and 7. Alkaline phosphatase activity assays were designed to measure the osteogenic differentiation of mesenchymal stem cell spheroids on Day 7. Alizarin Red S staining was performed to investigate the mineralization of cell spheroids on Days 7 and 14. Real-time polymerase chain reactions were used to measure the expression levels of RUNX2 and COL1A1 on Day 14. Western blot techniques were performed to identify the protein expression of Runt-related transcription factor 2 and type I collagen. Results: The control group’s mesenchymal stem cells displayed a spheroid shape. There was no noticeable change in morphology with the addition of CMTs at final concentrations of 0.001, 0.01, 0.1, and 1 μg/mL compared with the untreated (control) group. The application of CMTs did not induce a significant change in cell viability. The relative alkaline phosphatase activity values in the 0.001, 0.01, 0.1, and 1 μg/mL CMT groups were 114.4% ± 8.2%, 130.6% ± 25.3%, 87.8% ± 3.4%, and 92.1% ± 6.8%, respectively, considering a control of 100% (100.0% ± 17.9%). On Day 14, calcium deposits were clearly observed in each group. The relative values of Alizarin Red S staining in the 0.001, 0.01, 0.1, and 1 μg/mL CMT groups were 100.1% ± 8.9%, 105.9% ± 0.0%, 109.7% ± 19.1%, and 87.0% ± 40.9%, respectively, considering a control of 100% (100.0% ± 28.7%). The addition of CMT significantly increased RUNX2 expression in the 0.01 μg/mL group and COL1A1 in the 0.001 and 0.01 μg/mL groups. Normalization of protein expression showed that the addition of CMTs significantly increased type I collagen expression in the 0.001, 0.01, and 1 μg/mL groups. Conclusions: In conclusion, CMTs influence the osteogenic differentiation of bone-marrow-derived mesenchymal stem cells and the use of CMTs may positively influence the osteogenic differentiation of cell spheroids.

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Development of a device useful to reproducibly produce large quantities of viable and uniform stem cell spheroids with controlled diameters

Cited by 11 publications

References 55 publications

Bioprinting of Stem Cell Spheroids Followed by Post‐Printing Chondrogenic Differentiation for Cartilage Tissue Engineering

Bioprinting of Stem Cell Spheroids Followed by Post‐Printing Chondrogenic Differentiation for Cartilage Tissue Engineering

Effects of Enamel Matrix Derivative on Cell Spheroids Made of Stem Cells Obtained from the Gingiva on Osteogenic Differentiation

Assessment of the Impacts of Centipeda minima (L.) on Cell Viability, and Osteogenic Differentiation of Mesenchymal Stem Cell Spheroids

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