Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Summary

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Abstract

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

Introduction

The prevalence of joint disease leads to significant economic burdens due to the increasing number of disabled patients and the costs associated with their care. Hyaline cartilage is a connective avascular tissue with limited regenerative potential¹. Prolonged use of certain non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and chemotherapy or radiotherapy can further diminish cartilage's regenerative capacity, nearly eliminating its ability to heal². This makes it challenging to obtain autologous cartilage cells for cellular therapy.

The technology of three-dimensional (3D)....

Protocol

​The study was reviewed and approved by the Ethics Committee of the LOPUKHIN FRCC PCM (protocol No. 2019/02 of April 9, 2019). All donor samples were obtained in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants and/or their legal guardians.

NOTE: Maintain sterile technique throughout the protocol. Warm all culture media and solutions to 37 °C before applying them to cells or spheroids. Cultivate cells in a CO₂

Discussion

iPSCs represent a transformative tool in regenerative medicine, offering the potential to generate patient-specific chondrocytes for cartilage repair. Current protocols leverage directed differentiation through mesodermal pathways, with key signaling molecules like TGF-β and BMP-2 promoting chondrocytic lineage commitment. These methods aim to replicate embryonic cartilage development, enabling the production of extracellular matrix components such as collagen type II and aggrecan, essential for functional cartilage.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin solution	Thermo Fisher Scientific	25300-062
0.25% Trypsin solution	Thermo Fisher Scientific	25200-072
Advanced DMEM/F12 Eagle's medium	Thermo Fisher Scientific	12634028
Aggrecan Monoclonal Antibody	Invitrogen	AHP0022	Host: Mouse; Dilution: 1/500
Ascorbic acid	Sigma	A4544	50 μg/mL
B-27 supplement	Thermo Fisher Scientific	17504044	1x or 2%
Beta-mercaptoethanol	Serva	28625	90 mM
BMP2	Miltenyi biotec	130-110-922	10 ng/mL
Chir 99021	Miltenyi biotec	130-103-926	10 μM
COL1A1 (E6A8E) Monoclonal antibody	CellSignalling	39952	Host: Rabbit; Dilution: 1/800
COL2A1 (M2139) Monoclonal antibody	Santa Cruz	sc-52658	Host: Mouse; Dilution: 1/50
Collagenase type II solution	PanEco	P011-2	0.01%
DAPI (4',6-diamidino-2-phenylindole)	Sigma-Aldrich	D9542-5MG	1 μg/mL
DMEM medium w/o glutamine	PanEco	С420п
Fetal bovine serum	Thermo Fisher Scientific	10270106	10%
Hanks' solution	PanEco	Р020п
Hybris 8 medium	PanEco	С780Е/Ф780
Insulin-Transferrin-Selenium solution	PanEco	Ф065	1x solution has the following concentrations: Insulin: 10 µg/mL; Transferrin: 5.5 µg/mL; Selenium 5 ng/mL
L-alanyl-L-glutamine	Thermo Fisher Scientific	35050038	2 mM
Matrigel Matrix	BD	354277	300 μg/mL
Penicillin-Streptomycin solution	PanEco	А063п	100 U/mL
Retinoic acid	Miltenyi biotec	130-117-339	10 nM
Rho kinase inhibitor Y27632	Miltenyi biotec	130-103-922	10 mM
Secondary Antibody Goat anti-Mouse IgG (H+L) Cross-Adsorbed, Alexa Fluor 555	Thermo Fisher Scientific	A21422	Host: Goat; Dilution: 1/500
Secondary Antibody Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed, Alexa Fluor Plus 555	Thermo Fisher Scientific	А32732	Host: Goat; Dilution: 1/500
Sox9 (D8G8H) Monoclonal antibody	CellSignalling	82630	Host: Rb; Dilution: 1/400
TeSR-1 medium	STEMCELL technologies	85850
TGF-β1	Miltenyi biotec	130-095-067	10 ng/mL