Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs

Summary

This study presents a reproducible biofabrication workflow using adipose-derived stem cell spheroids and 3D-printed GelMA scaffolds. It demonstrates spatial control, fusion capacity, and quality control metrics, resulting in transplantable macroscale tissue constructs with potential applications in regenerative medicine.

Abstract

The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient transfer of biofabrication technologies. Rigorous biological standardization not only enhances reproducibility but also facilitates alignment with national and international regulatory frameworks, which are essential for the clinical translation of tissue engineering strategies. This study introduces a standardized biofabrication workflow comprising monolayer culture of hADSCs, spheroid formation, 3D printing of Gelatin Methacryloyl (GelMA) scaffolds, and spheroid bioassembly. Quality control metrics were integrated to ensure reproducibility. The 3D-printed GelMA scaffold was designed with two central pores, each capable of housing hundreds of spheroids. Printing fidelity was confirmed by comparing scaffold dimensions to the CAD model, with minor non-significant deviations. hADSCs formed uniform spheroids within 24 h in agarose micromolds. When seeded into scaffold pores, spheroids underwent continuous fusion over 72 h, forming macroscale constructs confirmed by confocal imaging. Scaffold design and printing accuracy ensured structural support, while spheroid uniformity enabled predictable formation of the construct. The fusion of spheroids within the scaffold pores yielded tissue constructs suitable for transplantation, with or without the scaffold, highlighting the versatility of this bioassembly approach. The findings underscore the pivotal role of biological standardization in advancing reproducible methodologies for the biofabrication of living tissue constructs, with promising implications for future clinical applications in regenerative medicine.

Introduction

Bioassembly and bioprinting are complementary biofabrication approaches in tissue engineering that focus on constructing complex tissues and organs at the macroscale using automated technologies^1,2,3,4.

Spheroids were incorporated into biofabrication as building blocks mainly for bioassembly approaches⁵. Spheroids are considered microtissues that reproduce interactions between cells and the surrounding tissue microenvironment in vivo. Among the cell sources used for spheroid format....

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of agarose micromolds and fabrication of GelMA 3D-printed scaffolds

1. Production of the 2% agarose micromold in 0.9% Sodium Chloride (NaCl) solution (see Figure 2A)
   NOTE: All chemical waste must be handled and disposed of in accordance with institutional chemical safety regulations and hazardous waste guidelines.
   1. Prepare a 0.9% NaCl solution in ultrapure water and autoclave for 30 min at 121 °C. Maintain under sterile conditions until use.<....

Results

Human adipose-derived stem cells (hASCs) cultured in a monolayer (Figure 2C) were successfully induced to form spheroids within 24 h after seeding into agarose micromolds (Figure 2D). The resulting spheroids exhibited a compact and well-defined morphology. Morphometric analysis revealed a mean diameter of 239.8 µm ± 87.0 µm at 24 h (Figure 2E) and a sphericity value close to 1 (0.947 ± 0.123) (Figure 2F.......

Discussion

The results shown in this study highlight the successful integration of quality control metrics into a standardized biofabrication workflow to ensure reproducibility. The use of 3D-printed scaffolds based on pre-established designs provides structural support for spheroid bioassembly^19,23. As a proof-of-concept, a solid scaffold was designed with two central porous regions, dimensioned to support high-density spheroid loading and guided bioassembly.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x Phosphate-Buffered Saline (PBS)	Prepared in the laboratory		-
593iCAN 3D Bioprinter, Model 593 iCan2X Printhead	593iCAN	-	-
Agarose	Sigma-Aldrich	16500-500	-
Ascorbic Acid	Sigma-Aldrich	A4544-100MG	-
Carl Zeiss Microscopy GmbH Primovert	Zeiss	-	415510-1101-000
Celldiscover	Zeiss	-	LSM 900 with Airyscan 2
Celltracker	Invitrogen	C7025	-
Centrifuge	Eppendorf	-	5702
Class II Biological Safety Cabinet	Streamline	-	Model SC2-4E3
CO2 incubator for cell culture	Thermo Fisher Scientific	-	Series 8000 WJ
Conical polypropylene nozzle 0.41 mm	593iCAN	-	-
Dulbecco’s Modified Eagle Medium Low Glucose	Sigma-Aldrich	D2902-10L	-
EDTA	Sigma-Aldrich	E9884	-
Fetal Bovine Serum (FBS)	Gibco	12657-029	-
Gelatin methacryloyl gel strength 300 g Bloom, degree of substitution 60%	Sigma-Aldrich	900622	Lot/Batch number: MKCS2673. Documentation for each batch, including the Safety Data Sheet (SDS) and the Certificate of Analysis (CoA), can be obtained from the manufacturer’s website.
GraphPad Prism 6.0 software	GraphPad Inc	-	-
Hot plate magnetic stirrer	IKA	Z671797	Model C-MAG HS7
Human serum albumin 100 mg/ml	FUJIFILM Irvine Scientific	9988	-
ITS (Insulin-transferrin-sodium selenite) supplement 100x	Sigma-Aldrich	I3146-5ML	-
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)	Sigma-Aldrich	900889
Luxoeo 4Z Stereozoom Microscope	Labomed	-	-
MicroTissues® 3D Petri Dish® micro-mold spheroids	Sigma-Aldrich	Z764019	-
Penicillin-streptomycin 100x	Sigma-Aldrich	15140122	-
Poietics™ human adipose derived stem cells (ADSC)	Lonza	PT-5006
Sodium Chloride (NaCl)	Sigma-Aldrich	71382-500G	-
Trypsin/EDTA	Gibco	27250018	-