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

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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Cells/colony motion of oral keratinocytes determined by non-invasive and quantitative measurement using optical flow predicts epithelial regenerative capacity

Scientific Reports ◽

10.1038/s41598-021-89073-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Emi Hoshikawa ◽

Taisuke Sato ◽

Kenta Haga ◽

Ayako Suzuki ◽

Ryota Kobayashi ◽

...

Keyword(s):

Optical Flow ◽

Cultured Cells ◽

Primary Cultures ◽

Culture Conditions ◽

Oral Keratinocytes ◽

Regenerative Capacity ◽

Non Invasive ◽

Quantitative Measurements ◽

Full Screen ◽

Quantitative Manner

AbstractCells/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 cells/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/h reflects cellular damages by experimental metabolic challenges although this value shall only apply in case of our culture system. Nonetheless, the motion index can be used as the threshold to determine the quality of cultured cells while it may be affected by any different culture conditions. Because the p1 cells/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

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Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.5.f651 ◽

1998 ◽

Vol 275 (5) ◽

pp. F651-F663 ◽

Cited By ~ 13

Author(s):

Isabelle Rubera ◽

Michel Tauc ◽

Michel Bidet ◽

Chantal Poujeol ◽

Béatrice Cuiller ◽

...

Keyword(s):

Epithelial Cells ◽

Cultured Cells ◽

Primary Cultures ◽

Physiological Role ◽

Cell Types ◽

Patch Clamp Technique ◽

Chloride Currents ◽

Hypotonic Stress ◽

Quantitative Measurements ◽

Whole Cell Patch Clamp

Cl− conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl− conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl−currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl− currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl− currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl− current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl− pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

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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

Journal of Tissue Engineering ◽

10.1177/2041731419881528 ◽

2019 ◽

Vol 10 ◽

pp. 204173141988152 ◽

Cited By ~ 1

Author(s):

Emi Hoshikawa ◽

Taisuke Sato ◽

Yoshitaka Kimori ◽

Ayako Suzuki ◽

Kenta Haga ◽

...

Keyword(s):

Quality Control ◽

Image Analysis ◽

Regenerative Medicine ◽

Cultured Cells ◽

Time Lapse ◽

Proliferative Capacity ◽

Oral Keratinocytes ◽

Positive Linear Correlation ◽

Consecutive Time ◽

Cell Colony

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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Carbohydrate metabolism by primary cultures of rabbit proximal tubules

AJP Cell Physiology ◽

10.1152/ajpcell.1989.256.3.c532 ◽

1989 ◽

Vol 256 (3) ◽

pp. C532-C539 ◽

Cited By ~ 37

Author(s):

M. J. Tang ◽

K. R. Suresh ◽

R. L. Tannen

Keyword(s):

Pyruvate Kinase ◽

Phosphoenolpyruvate Carboxykinase ◽

Cultured Cells ◽

Primary Cultures ◽

Lactate Production ◽

Proximal Tubules ◽

Glycolytic Metabolism ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

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Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Endocrinology ◽

10.1210/endo.139.1.5677 ◽

1998 ◽

Vol 139 (1) ◽

pp. 35-43 ◽

Cited By ~ 29

Author(s):

Thomas O. Carpenter ◽

Kathleen C. Moltz ◽

Bruce Ellis ◽

Monica Andreoli ◽

Thomas L. McCarthy ◽

...

Keyword(s):

Messenger Rna ◽

Cultured Cells ◽

Primary Cultures ◽

Specific Effect ◽

Continuous Exposure ◽

Primary Osteoblast ◽

Characteristic Features ◽

Species Specific ◽

Osteocalcin Production

Abstract Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

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Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma

International Journal of Gynecological Cancer ◽

10.1136/ijgc-00009577-200407000-00006 ◽

2004 ◽

Vol 14 (4) ◽

pp. 607-615 ◽

Cited By ~ 1

Author(s):

P. Kornblith ◽

R. L. Ochs ◽

A. Wells ◽

M. J. Gabrin ◽

J. Piwowar ◽

...

Keyword(s):

Ovarian Cancer ◽

Cultured Cells ◽

Primary Cultures ◽

Human Tumor ◽

Clinical Results ◽

Chemotherapeutic Agents ◽

Primary Ovarian Cancer ◽

In Vitro Effects ◽

Therapeutic Decision Making

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

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Cadmium inhibits glucose uptake in primary cultures of mouse cortical tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.1990.258.6.f1625 ◽

1990 ◽

Vol 258 (6) ◽

pp. F1625-F1633 ◽

Cited By ~ 4

Author(s):

S. S. Blumenthal ◽

D. L. Lewand ◽

M. A. Buday ◽

J. G. Kleinman ◽

S. K. Krezoski ◽

...

Keyword(s):

Amino Acid ◽

Glucose Uptake ◽

Cultured Cells ◽

Primary Cultures ◽

Defined Medium ◽

Isobutyric Acid ◽

Cell Permeability ◽

Dependent Manner ◽

Lactate Dehydrogenase Activity ◽

Tubule Cells

We studied the effect of cadmium (Cd2+) on transport of alpha-methylglucoside in primary cultures of mouse kidney cortical tubule cells grown in defined medium. When cultured cells were exposed to Cd2+ concentrations from 0 to 6 microM for 24 h, uptake of alpha-methylglucoside was inhibited in a dose-dependent manner by up to 50%. By contrast, acute exposure of the cells to 7 microM Cd2+ for 60 min did not inhibit alpha-methylglucoside uptake. Increasing Cd2+ concentrations progressively decreased the Vmax of Na(+)-dependent glucose cotransport but not the Km for glucose. Cell ATP/ADP ratios of unexposed monolayers and of cells exposed to 4.5 microM Cd2+ for 24 h were 5.0 and 4.9, respectively (n = 3). Intracellular volume, lactate dehydrogenase activity, and cell Na+ and K+ concentrations were unaltered even after 24 h of exposure to 7 microM Cd2+. Untreated and Cd2+-treated monolayers preloaded with alpha-methylglucoside released the sugar analogue into the medium at nearly identical rates, indicating that Cd2+ did not alter cell permeability to glucose. Uptake of the amino acid analogue alpha-(methylamino)isobutyric acid was not affected by prior Cd2+ exposure. Whereas cell DNA content declined in Cd2(+)-exposed plates, both Na(+)-glucose and Na(+)-amino acid cotransport were enhanced at lower cell densities. Protein and DNA synthesis, estimated, respectively, by incorporation of [3H]leucine and [3H]thymidine into acid-insoluble material, were not significantly affected at 6 microM Cd2+. We conclude that after a lag time Cd2+ selectively inhibits renal Na(+)-dependent glucose transport despite an unchanged gradient for Na+ across the cell membrane.

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In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid

Biochemical Journal ◽

10.1042/bj3170827 ◽

1996 ◽

Vol 317 (3) ◽

pp. 827-833 ◽

Cited By ~ 83

Author(s):

Pere PUIGSERVER ◽

Francisca VÁZQUEZ ◽

María L. BONET ◽

Catalina PICÓ ◽

Andreu PALOU

Keyword(s):

Retinoic Acid ◽

Cultured Cells ◽

Uncoupling Protein ◽

Primary Cultures ◽

Brown Adipocyte ◽

Brown Adipose ◽

Adipocyte Cell ◽

Differentiation Stage

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.

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5-HT2B receptors are expressed on astrocytes from brain and in culture and are a chronic target for all five conventional ‘serotonin-specific reuptake inhibitors’

Neuron Glia Biology ◽

10.1017/s1740925x10000141 ◽

2010 ◽

Vol 6 (2) ◽

pp. 113-125 ◽

Cited By ~ 33

Author(s):

Shiquen Zhang ◽

Baoman Li ◽

Ditte Lovatt ◽

Junnan Xu ◽

Dan Song ◽

...

Keyword(s):

Protein Expression ◽

Cell Sorting ◽

Cultured Cells ◽

Primary Cultures ◽

Rt Pcr ◽

Chain Reaction ◽

Calcium Dependent ◽

Mrna And Protein Expression ◽

Polymerase Chain ◽

Well Differentiated

In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the ‘serotonin-specific reuptake inhibitor’ (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 μM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1–0.5 μM; these are therapeutically relevant concentrations.

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