Noninvasive measurement of cell/colony motion using image analysis methods to evaluate the proliferative capacity of oral keratinocytes as a tool for quality control in regenerative medicine

Image-based cell/colony analyses offer promising solutions to compensate for the lack of quality control (QC) tools for noninvasive monitoring of cultured cells, a regulatory challenge in regenerative medicine. Here, the feasibility of two image analysis algorithms, optical flow and normalised cross-correlation, to noninvasively measure cell/colony motion in human primary oral keratinocytes for screening the proliferative capacity of cells in the early phases of cell culture were examined. We applied our software to movies converted from 96 consecutive time-lapse phase-contrast images of an oral keratinocyte culture. After segmenting the growing colonies, two indices were calculated based on each algorithm. The correlation between each index of the colonies and their proliferative capacity was evaluated. The software was able to assess cell/colony motion noninvasively, and each index reflected the observed cell kinetics. A positive linear correlation was found between cell/colony motion and proliferative capacity, indicating that both algorithms are potential tools for QC.

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Cell/Colony Motion of Oral Keratinocytes Determined by Non-Invasive and Quantitative Measurement Using Optical Flow Predicts Epithelial Regenerative Capacity

10.21203/rs.3.rs-236508/v1 ◽

2021 ◽

Author(s):

Emi Hoshikawa ◽

Taisuke Sato ◽

Kenta Haga ◽

Ayako Suzuki ◽

Ryota Kobayashi ◽

...

Keyword(s):

Optical Flow ◽

Cultured Cells ◽

Primary Cultures ◽

Oral Keratinocytes ◽

Regenerative Capacity ◽

Non Invasive ◽

Quantitative Measurements ◽

Full Screen ◽

Quantitative Manner ◽

Cell Colony

Abstract Cell/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cell/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage (passage 1 (p1)) cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 μm/hour reflects cellular damages by experimental metabolic challenges and can be used as the threshold to determine the quality of cultured cells. Because the p1 cell/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

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Binary and mixed population biofilms: Time-lapse image analysis and disinfection with biocides

Journal of Industrial Microbiology & Biotechnology ◽

10.1038/sj/jim/7000318 ◽

2002 ◽

Vol 29 (6) ◽

pp. 331-338

Author(s):

K T Elvers ◽

K Leeming ◽

H M Lappin-Scott

Keyword(s):

Image Analysis ◽

Time Lapse ◽

Mixed Population

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Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

Scientific Reports ◽

10.1038/s41598-021-93607-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shojiro Katoh ◽

Atsuki Fujimaru ◽

Masaru Iwasaki ◽

Hiroshi Yoshioka ◽

Rajappa Senthilkumar ◽

...

Keyword(s):

Regenerative Medicine ◽

Replicative Senescence ◽

Cultured Cells ◽

Three Dimensional ◽

Human Chondrocytes ◽

Cartilage Tissue ◽

Optimal Method ◽

Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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Automatic system of quality control of hybrid circuits by image analysis

[1992] Proceedings. 11th IAPR International Conference on Pattern Recognition ◽

10.1109/icpr.1992.201593 ◽

2003 ◽

Author(s):

K. Chehdi ◽

M. Corazza

Keyword(s):

Quality Control ◽

Image Analysis ◽

Automatic System

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34 LIFESPAN AND CHROMOSOMAL STABILITY OF BOVINE AND PORCINE FETAL FIBROBLAST CELLS CULTURED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab34 ◽

2005 ◽

Vol 17 (2) ◽

pp. 167 ◽

Cited By ~ 2

Author(s):

A.M. Giraldo ◽

J.W. Lynn ◽

C.E. Pope ◽

R.A. Godke ◽

K.R. Bondioli

Keyword(s):

Cell Lines ◽

Cultured Cells ◽

Culture Conditions ◽

Cycle Duration ◽

Fibroblast Cells ◽

Proliferative Capacity ◽

Chromosomal Stability ◽

Fetal Fibroblasts ◽

Fetal Bovine

The low efficiency of nuclear transfer (NT) has been related to factors such as mitochondria heteroplasmy, failure of genomic activation, and asynchrony between the donor karyoplast and recipient cytoplast. Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. It is known that suboptimal culture conditions can induce chromosomal abnormalities, and the use of aneuploid donor cells during NT can lead to a high incidence of abnormal cloned embryos (Giraldo et al. 2004 Reprod. Fertil. Dev. 16, 124 abst). The purpose of this study was to determine the lifespan and chromosomal stability of bovine and porcine fetal cells. Four bovine and four porcine fibroblast cells lines were established from 50-day and 40-day fetuses, respectively. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in 5% CO2. Each cell line was passaged to senescence. Total population doublings (PDs) and cell cycle duration were calculated. To determine the chromosome numbers at different PDs, cells were synchronized in metaphase, fixed, and stained. ANOVA and chi-square tests were used to analyze differences in PDs and proportion of aneuploid cells between cell lines, respectively (P < 0.05). The results show that proliferative capacity was not different between cell lines derived from the same species. Cell lines derived from bovine and porcine fetuses had different in vitro lifespans (33 PDs vs. 42 PDs, respectively; P < 0.05). The mean length of the cell cycles for both bovine and porcine fetal fibroblasts was ∼28 h. The percentage of aneupliod cells in both bovine and porcine fetal cell lines increased progressively with duration of culture (see Table) and was high throughout the study. The proliferative capacity of cultured cells was similar within individuals of the same species, but growth characteristics differed between fetal bovine and porcine cell lines. The progressive increase of aneuploid cells could be due to suboptimal culture conditions or unusual chromosome instability in the particular fetuses used. These data demonstrate the importance of determining chromosome content and the use of cells at early passages to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of NT.

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