Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

Maimonah-Eissa Al-Masawa,Kien-Hui Chua,Wan Safwani Wan Kamarul Zaman

doi:10.1038/s41598-020-78395-y

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

Maimonah-Eissa Al-Masawa, Kien-Hui Chua + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-78395-y

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Abstract

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Highlights

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering
We evaluated the growth factor cocktail for its potential to induce DNA damage, TP53 mutation, aneuploidy, cell cycle and tumour suppressor genes’ expression (p53, p21 and pRB) in human nasal septal chondrocytes (hNSCs) culture-expanded for six passages
We have examined the possibility of any tumorigenic transformation of the growth factor cocktail-manipulated hNSCs in vivo in a nude mouse model

Summary

Introduction

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works. Employed to improve chondrocyte culture condition is the supplementation of culture medium with cartilaginous growth factors[12] The selection of these growth factors is based on the understanding of their vital role in the regulation of cartilage developmental p rocesses[13]. The use of growth factors has allowed for the formulation of low percentage autologous serum or even serum-free media, solving the controversy over standardization and use of xenogeneic serum in c ulture[19] This extensive ex vivo manipulation of chondrocytes using exogenous growth factors raises the issue of their clinical biosafety. While cell products are usually tested for identity, viability, sterility, and endotoxin level; there are still no standardised comprehensive preclinical protocols to check for genomic instability, small molecular aberrations, or tumorigenic transformation p otential[20,23]

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