Development of human fibroblast spheroids with hanging-drop inverted plates
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2023
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LATIN-AMERICAN CONGRESS OF ARTIFICIAL ORGANS AND BIOMATERIALS, 12th
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Introduction and objective: 2D cultures have limitations in cell growth. 3D cultures, on the other hand, have become a
valuable and powerful tool for biomedical research in recent decades. Due to their resemblance to living systems and
cellular interactions, this type of culture can be developed using various methodologies, including nanoparticles,
hydrogels, and layers of agarose, among others. Considering the need for testing and validating new molecules and
effective therapies for treating various diseases, the objective of this study is to standardize a 3D human fibroblast
culture model.
Methodology: HF002-J, human fibroblast cells, were cultured at 37ºC in a humid atmosphere containing 5% CO2,
maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics. When reaching 60-
70% confluence, the cells were detached using a 0.05% trypsin solution. Spheroids were prepared using the hangingdrop technique adapted from [1] 440 µL of medium containing cell variations ranging from 2 × 103 to 6
× 104 cells per well of a 96-well plate were deposited, generating a positive meniscus. The plate was inverted and
incubated as described.
Results and discussion: The present study aimed to evaluate the development of cellular spheroids after 4 days of
culture using different cell preparations. Our results demonstrated that the preparations used produced compact
spheroids, characterized by homogeneous sizes in the range of 500 to 1000 μm. When analysing the images obtained
by wide-field fluorescence microscopy, we observed that the proportions of unviable cells labelled with fluorophores
varied significantly according to the initial number of cells used in the preparations. Notably, increasing the initial
number of cells resulted in a proportional increase in the number of non-viable cells present in the formed spheroids.
These results suggest that the initial cell density can affect the development and viability of the formed spheroids. It is
possible that too high cell density led to greater competition for nutrients and space, resulting in greater cell mortality
and less viable spheroids.
Conclusions: Based on the results obtained, it was possible to develop an initial prototype of spheroids from human
fibroblast cells that can resemble tissues in vivo due to their cellular interactions, thus providing a new tool for the study
of drugs and treatments.
Como referenciar
RODRIGUES, ALEX A.; SAMPAIO, MARLOS C.; SANTOS, ESTHER C. dos; PRUDENTE, SULEYNA R.; LIMA, MAYELLE M.P.; SILVA, GIOVANA D. da; MATHOR, MONICA B.; VIEIRA, DANIEL P. Development of human fibroblast spheroids with hanging-drop inverted plates. In: LATIN-AMERICAN CONGRESS OF ARTIFICIAL ORGANS AND BIOMATERIALS, 12th, December 12-15, 2023, Mar del Plata, Argentina. Abstract... p. 213-213. Disponível em: https://repositorio.ipen.br/handle/123456789/47936. Acesso em: 05 Mar 2025.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.