Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Summary

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Abstract

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 µm. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and >95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Introduction

Tissue spheroids are 3D micro-tissues formed by cell suspensions that undergo self-assembly without external forces¹. These spheroids have been widely used in biofabrication protocols due to their resemblance with key features of the human physiological system^2,3. Tissue spheroids provide more similar metabolism, cytoskeleton dynamics, cell viability, and metabolic and secretion activity than traditional monolayer cell culture¹. Due to their fusion capability, they can also be used as building blocks (e.g., bioprinting protocols) to form complex tissue-engineered....

Protocol

The L929 cell line, mouse fibroblasts, was used for the present study. The stamp-like 3D printed biodevice was obtained from a commercial source (see Table of Materials). Good cell culture practice and sterile techniques were followed throughout the protocol. The fabricated device was sterilized by wiping it with 70% alcohol and exposing it to UV light for 15 min. The cell culture media and solutions were warmed to 37 °C before contacting with the cells or tissue spheroids. A schematic representatio.......

Discussion

The present protocol describes a simple, fast, and inexpensive method for the large-scale production of tissue spheroids. A stamp-like 3D printed device was used as a master mold, which generated up to 4,716 spheroids per 6-well plate. It has been shown that cells cultivated as spheroids have more realistic biological responses that closely resemble the in vivo environment¹. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more pred.......

Materials

Name	Company	Catalog Number	Comments
6 well plate	Merck	CLS3516
Agarose	Promega	V3121
Biodevice	Bioedtech
Biological Safety Cabinet	ThermoFisher	51029701
Centrifugue	ThermoFisher	75004031
Corning 50 mL centrifuge tubes	Merck	CLS430829-500EA
Corning cell culture flasks surface area 75 cm2	Merck	CLS430641
Draft Resin	FormLabs	FLDRBL01
Dulbecco′s Modified Eagle′s Medium - low glucose	Merck	D6046
Fetal Bovine Serum (FBS)	ThermoFisher	16000044
Form 2	FormLabs
Incubator	ThermoFisher	51033782
L929 cell lines	Stablished in the lab
Penicillin and Streptomycin (PS)	ThermoFisher	15140122
Phosphate-Buffered Saline (PBS)	Merck	806552
Trypsin with EDTA	Merck	T3924