scholarly journals The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Nikko Brix ◽  
Daniel Samaga ◽  
Roman Hennel ◽  
Katharina Gehr ◽  
Horst Zitzelsberger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Survival Data ◽  
State Of The Art ◽  
Clonogenic Assay ◽  
Clonogenic Survival ◽  
Growth Behavior ◽  
Plating Efficiency ◽  
Current State ◽  
Clonogenic Growth ◽  
Cellular Cooperation

Abstract Background The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. Methods A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. Results For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. Conclusions Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

scholarly journals The Clonogenic Assay: Robustness of Plating-Efficiency-Based Analysis is Strongly Compromised by Cellular Cooperation

2020 ◽  
Author(s):  
Nikko Brix ◽  
Daniel Samaga ◽  
Roman Hennel ◽  
Katharina Gehr ◽  
Horst Zitzelsberger ◽  
...  
Keyword(s):  
Cell Lines ◽  
State Of The Art ◽  
Clonogenic Assay ◽  
Clonogenic Survival ◽  
Growth Behavior ◽  
Plating Efficiency ◽  
Mathematical Approach ◽  
Current State ◽  
Clonogenic Growth ◽  
Cellular Cooperation

Abstract Background:The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained.Methods:A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto‑/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data.Results:For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common.Conclusions:Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

scholarly journals Over-Production of Therapeutic Growth Factors for Cartilage Regeneration by Protein Production Platforms and Protein Packaging Cell Lines: A Narrative Review of the Current State-of-the-Art

2019 ◽  
Author(s):  
Ali Mobasheri ◽  
Pablo Martín-Vasallo
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Protein Production ◽  
State Of The Art ◽  
Cartilage Tissue ◽  
Cartilage Defects ◽  
Scale Production ◽  
Packaging Cell ◽  
Current State ◽  
Mammalian Protein

This article focuses on the current state-of-the-art in the area of cellular and molecular biotechnology for over-production of clinically relevant therapeutic growth factors and how the technology can be used for the treatment of osteoarthritis (OA). Transfected and irradiated protein packaging cell lines may be used as “cellular factories” for large-scale production of therapeutic proteins and pro-anabolic growth factors, particularly in the context of cartilage matrix regeneration. We discuss the potential for new innovations in regenerative medicine for degenerative diseases of synovial joints using mammalian protein production platforms, specifically protein packaging cell lines, for over-producing growth factors for cartilage tissue regeneration and give recent examples. Mammalian protein production platforms that incorporate protein packaging cell lines are superior to bacterial expression systems and are likely to have a significant impact on the development of new biological therapies for treating focal cartilage defects and more generally for the treatment of degenerative joint diseases such as OA.

scholarly journals Comparison of Clonogenic Survival Data Obtained by Pre- and Post-Irradiation Methods

2020 ◽  
Vol 10 (4) ◽  
pp. 171
Author(s):  
Takahiro Oike ◽  
Yuka Hirota ◽  
Narisa Dewi Maulany Darwis ◽  
Atsushi Shibata ◽  
Tatsuya Ohno
Keyword(s):  
Survival Data ◽  
Gold Standard ◽  
Clonogenic Assay ◽  
Clonogenic Survival ◽  
Integrated Analysis ◽  
Common Cancer ◽  
Plating Method ◽  
Biological Insight ◽  
Cell Plating

Clonogenic assays are the gold standard to measure in vitro radiosensitivity, which use two cell plating methods, before or after irradiation (IR). However, the effect of the plating method on the experimental outcome remains unelucidated. By using common cancer cell lines, here we demonstrate that pre-IR and post-IR plating methods have a negligible effect on the clonogenic assay-derived photon sensitivity as assessed by SF2, SF4, SF6, SF8, D10, or D50 (N.B. SFx indicates the survival at X Gy; Dx indicates the dose providing X% survival). These data provide important biological insight that supports inter-study comparison and integrated analysis of published clonogenic assay data regardless of the plating method used.

Behavioral Genetics: Concepts for Research and Practice in Language Development and Disorders

1995 ◽  
Vol 38 (5) ◽  
pp. 1126-1142 ◽  
Author(s):  
Jeffrey W. Gilger
Keyword(s):  
Language Development ◽  
Behavioral Genetics ◽  
State Of The Art ◽  
Genetic Research ◽  
Great Promise ◽  
Behavioral Genetic ◽  
Fine Grained ◽  
Future Goals ◽  
Current State ◽  
Research Designs

This paper is an introduction to behavioral genetics for researchers and practioners in language development and disorders. The specific aims are to illustrate some essential concepts and to show how behavioral genetic research can be applied to the language sciences. Past genetic research on language-related traits has tended to focus on simple etiology (i.e., the heritability or familiality of language skills). The current state of the art, however, suggests that great promise lies in addressing more complex questions through behavioral genetic paradigms. In terms of future goals it is suggested that: (a) more behavioral genetic work of all types should be done—including replications and expansions of preliminary studies already in print; (b) work should focus on fine-grained, theory-based phenotypes with research designs that can address complex questions in language development; and (c) work in this area should utilize a variety of samples and methods (e.g., twin and family samples, heritability and segregation analyses, linkage and association tests, etc.).

Schizophrenia Research: Current State of the Art

1976 ◽  
Vol 21 (7) ◽  
pp. 497-498
Author(s):  
STANLEY GRAND
Keyword(s):  
State Of The Art ◽  
Current State

Umbral Calculus

10.37236/24 ◽  
2002 ◽  
Vol 1000 ◽  
Author(s):  
A. Di Bucchianico ◽  
D. Loeb
Keyword(s):  
19Th Century ◽  
Finite Differences ◽  
State Of The Art ◽  
Umbral Calculus ◽  
Current State ◽  
Logical Foundation ◽  
Complete Bibliography ◽  
The 19Th Century ◽  
Mathematical Literature

We survey the mathematical literature on umbral calculus (otherwise known as the calculus of finite differences) from its roots in the 19th century (and earlier) as a set of “magic rules” for lowering and raising indices, through its rebirth in the 1970’s as Rota’s school set it on a firm logical foundation using operator methods, to the current state of the art with numerous generalizations and applications. The survey itself is complemented by a fairly complete bibliography (over 500 references) which we expect to update regularly.

The Receptor-Dependent QSAR Paradigm: An Overview of the Current State of the Art

2009 ◽  
Vol 5 (4) ◽  
pp. 359-366 ◽  
Author(s):  
Osvaldo Santos-Filho ◽  
Anton Hopfinger ◽  
Artem Cherkasov ◽  
Ricardo de Alencastro
Keyword(s):  
State Of The Art ◽  
Current State

scholarly journals Smoothing additive manufactured parts using ns-pulsed laser radiation

2021 ◽  
Author(s):  
Florian Kuisat ◽  
Fernando Lasagni ◽  
Andrés Fabián Lasagni
Keyword(s):  
Surface Roughness ◽  
Mechanical Performance ◽  
State Of The Art ◽  
Pulsed Laser ◽  
Industrial Applications ◽  
Laser Source ◽  
Pulsed Laser Radiation ◽  
Current State ◽  
The Impact

AbstractIt is well known that the surface topography of a part can affect its mechanical performance, which is typical in additive manufacturing. In this context, we report about the surface modification of additive manufactured components made of Titanium 64 (Ti64) and Scalmalloy®, using a pulsed laser, with the aim of reducing their surface roughness. In our experiments, a nanosecond-pulsed infrared laser source with variable pulse durations between 8 and 200 ns was applied. The impact of varying a large number of parameters on the surface quality of the smoothed areas was investigated. The results demonstrated a reduction of surface roughness Sa by more than 80% for Titanium 64 and by 65% for Scalmalloy® samples. This allows to extend the applicability of additive manufactured components beyond the current state of the art and break new ground for the application in various industrial applications such as in aerospace.

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