Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Summary

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Abstract

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.

Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.

The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Introduction

Spheroids are considered a scaffold-free approach in tissue engineering. ASCs are capable of forming spheroids by the self-assembly process. The spheroid's 3D microarchitecture increases the regenerative potential of ASCs, including the differentiation capacity into multiple lineages^1,2,3. This research group has been working with ASC spheroids for cartilage and bone tissue engineering^4,5,6. More importantly, spheroids are considered building-blocks in the biofabrication of tissu....

Protocol

The ASCs used in this study were previously isolated from healthy human donors and cryopreserved as described¹⁴ according to the Research Ethics Committee of Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Brazil (25818719.4.0000.5257). See Table of Materials for details regarding all the materials and equipment used in this study.

1. Trypsinization of ASC monolayer at passage three

1. Open the tissue culture 175 cm² flask containing the monolayer of ASCs at 80% confluence and discard the supernatant.
2. Add 7 mL of phospha....

Results

The automatic pipette system can seed the ASC cell suspension into 12 wells of one 12-well plate in 15 min. Using the 81 micromolded non-adherent hydrogel will produce 972 spheroids at the end of the protocol. Using the 256 micromolded non-adherent hydrogel will produce 3,072 spheroids at the end of the protocol. ASC spheroids were analyzed for the homogeneity of their size and shape. ASC spheroids from micromolds with 81 recessions showed homogeneous diameter during the culture period in contrast to ASC spheroids from m.......

Discussion

This paper presents the large-scale generation of ASC spheroids using an automated pipette system. The critical step of the protocol is to precisely set up the software to ensure the correct volume of cell suspension, speed, and distance for pipetting. The parameters described in the protocol were determined after a number of trials to optimize the dispensing of the ASC cell suspension into the wells of 12-well plates containing the micromolded, non-adherent hydrogels. The optimization was evaluated by measuring the diam.......

Materials

Name	Company	Catalog Number	Comments
12-well plastic plate	Corning	3512
50 mL centrifuge tube	Corning	CLS430828
EpMotion 5070	Eppendorf	5070000282
epT.I.P.S. Motion	Eppendorf	30015231
ethylenediaminetetraacetic acid (EDTA)	Invitrogen	15576028
fetal bovine serum (FBS)	Gibco	10082147
Low Glucose Dulbecco's Modified Eagle Medium (DMEM LOW)	Gibco	31600034
MicroTissues 3D Petri Dish micro-mold spheroids - 16 x 16 array	Sigma	Z764000
MicroTissues 3D Petri Dish micro-mold spheroids - 9 x 9 array	Sigma	Z764019
phosphate saline buffer (PBS)	Sigma	806552
sodium chloride (NaCl)	Sigma	S8776
tissue culture flask	Corning	430720U
trypan	Lonza	17-942E
trypsin	Gibco	27250018
ultrapure agarose	Invitrogen	16500100