Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array

Wallenborn, Marco; Petters, O; Rudolf, Despina; Hantmann, Helene; Richter, Margaret A.; Ahnert, Peter; Rohani, Leili; Jj, Smink; Gc, Bulwin; Krupp, Wolfgang; Rm, Schulz; Holland, Heidrun

doi:10.22203/ecm.v035a16

Cited by 9 publications

(15 citation statements)

References 14 publications

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“…While some previous studies demonstrated genetic instability had taken place in a long term ex vivo culture (with no growth factors) due to replicative stress 28 , 29 , others suggested long term ex vivo expansion to be safe 30 , 33 . Consequently, we were curious to investigate the effect of the stress growth factors might induce on human chondrocytes in long term culture- especially that studies on the biosafety of in vitro manipulated chondrocytes with growth factors are still limited.…”

Section: Discussionmentioning

confidence: 99%

“…Lately, there has been a growing concern regarding the biosafety of the ex vivo expanded cells. Contradictory reports on the incidence of cytogenetic alterations at progressive culture times 28 – 30 , 33 , 60 may have arisen from the fact that the employed techniques greatly differed in their sensitivity. Moreover, the vast majority of current techniques—including the techniques used in this study—are unable to sense anomalies arise in below 10% of the examined cells 22 .…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it is vital that the risk of tumorigenic transformation due to growth factors’ use in vitro be examined. With increasing reports on the incidence of cytogenetic aberrations during cell ex vivo manipulation 28 , only a few attempts were made to investigate genetic alterations and tumorigenicity in chondrocytes induced by growth factors 29 . Therefore, in the current study, we investigated the biosafety of in vitro expanded human nasal septal chondrocytes (hNSCs) in growth factor cocktail supplemented culture medium.…”

Section: Introductionmentioning

confidence: 99%

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

Al-Masawa

Zaman

Chua

2020

Sci Rep

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Al-Masawa

Zaman

Chua

2020

Sci Rep

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“…However, we decided to stop cell culture in passage P2, when AF and NP cells reached a cPDL of 9.4 ± 0.8 and 8.5 ± 0.8 on average, respectively, to avoid a bias by general cell culture effects. It is known from other cartilage cells that the genetic stability is altered, when cells are cultured higher than a cPDL of 10 [ 46 ].…”

Section: Discussionmentioning

confidence: 99%

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells

Schubert¹,

Smink²,

Pumberger³

et al. 2018

J Orthop Surg Res

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BackgroundThe lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell’s behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture.MethodsHuman AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) or Ham’s F-12 medium (Ham’s F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation.ResultsAF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham’s F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham’s F-12.ConclusionThe impact of the different media itself on disc cell’s behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

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“…This aims to identify potential adverse outcomes and then design the pivotal studies to examine their likelihood. For MSCs there are number of common risks to consider including but not limited to: tumorgenicity, immunogenicity, chromosomal instability, and unsafe biodistribution profiles [ 22 , 135–137 ]. From a regulatory perspective, it will be required to justify the model's appropriateness, both in terms of simulated condition and duration.…”

Section: Pre-clinical Testing Of Cellular Productsmentioning

confidence: 99%

Hurdles to uptake of mesenchymal stem cells and their progenitors in therapeutic products

Childs

Reid

Salmerón‐Sánchez

et al. 2020

Biochemical Journal

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Twenty-five years have passed since the first clinical trial utilising mesenchymal stomal/stem cells (MSCs) in 1995. In this time academic research has grown our understanding of MSC biochemistry and our ability to manipulate these cells in vitro using chemical, biomaterial, and mechanical methods. Research has been emboldened by the promise that MSCs can treat illness and repair damaged tissues through their capacity for immunomodulation and differentiation. Since 1995, 31 therapeutic products containing MSCs and/or progenitors have reached the market with the level of in vitro manipulation varying significantly. In this review, we summarise existing therapeutic products containing MSCs or mesenchymal progenitor cells and examine the challenges faced when developing new therapeutic products. Successful progression to clinical trial, and ultimately market, requires a thorough understanding of these hurdles at the earliest stages of in vitro pre-clinical development. It is beneficial to understand the health economic benefit for a new product and the reimbursement potential within various healthcare systems. Pre-clinical studies should be selected to demonstrate efficacy and safety for the specific clinical indication in humans, to avoid duplication of effort and minimise animal usage. Early consideration should also be given to manufacturing: how cell manipulation methods will integrate into highly controlled workflows and how they will be scaled up to produce clinically relevant quantities of cells. Finally, we summarise the main regulatory pathways for these clinical products, which can help shape early therapeutic design and testing.

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Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array

Cited by 9 publications

References 14 publications

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

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

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells

Hurdles to uptake of mesenchymal stem cells and their progenitors in therapeutic products

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