Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy

AbstractThe population decline in the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we detailed a method to establish fibroblast cell cultures from a female common hippopotamus fetus in this study. By combining the classic tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Characterization of the fibroblast cultures was carried out using different techniques. In short, neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 23.9 h according to the growth curve. Karyotyping based on Giemsa staining showed that cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. Mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with the Hippopotamus amphibius complete mitochondrial DNA sequence registered in GenBank, confirming the cells were derived from a common hippopotamus. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4 and Vimentin. In conclusion, we isolated and characterized a new fibroblast cell culture from a common hippopotamus skin sample and the cryopreserved cells could be useful genetic materials for the future research.

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Establishment and characterization of fibroblast cell line of the Songliao Black pig

Canadian Journal of Animal Science ◽

10.4141/cjas09013 ◽

2009 ◽

Vol 89 (4) ◽

pp. 463-466

Author(s):

X Liu ◽

L -F Li ◽

X Li ◽

Y Ma ◽

W Guan

Keyword(s):

Cell Line ◽

Karyotype Analysis ◽

Fibroblast Cell ◽

Dna Fingerprint ◽

Population Doubling ◽

Population Doubling Time ◽

Fibroblast Cell Line ◽

Primary Explants ◽

Genomic Studies

The Songliao Black pig was the first lean-meat maternal breed bred in China. To preserve genetic diversity of this breed, we designed a procedure to use ear marginal tissue of one male pig to establish a fibroblast cell line (designated SBPF18), which was subsequently characterized according to the population doubling time, karyotype analysis, isoenzyme assay, DNA fingerprint and sex determination, meanwhile microbial contamination assays were conducted to demonstrate the cell line was contamination-free. This method is suitable for establishing the fibroblast cell line from small amounts of tissue, and could further facilitate genomic studies and genetic modification.Key words: Songliao Black pig, fibroblast cell line, primary explants technique, cryogenic preservation

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Establishment, Culture, and Characterization of Guinea Pig Fetal Fibroblast Cell

Veterinary Medicine International ◽

10.1155/2014/510328 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 10

Author(s):

Davood Mehrabani ◽

Reza Mahboobi ◽

Mehdi Dianatpour ◽

Shahrokh Zare ◽

Amin Tamadon ◽

...

Keyword(s):

Guinea Pig ◽

Culture Method ◽

Fibroblast Cell ◽

Biological Evaluation ◽

Population Doubling ◽

Population Doubling Time ◽

Average Cell ◽

Fetal Fibroblast ◽

Cell Counts ◽

Fetal Fibroblast Cell

Establishment of Guinea pig fetal fibroblast cells and their biological evaluation before and after cryopreservation were the main purposes of this study. After determination of the proper age of pregnancy by ultrasonography, 30 days old fetuses of Guinea pigs were recovered. Their skins were cut into small pieces (1 mm2) and were cultured. When reaching 80–90% confluence, the cells were passaged. Cells of the second and eighth passages were cultured in 24-well plates (4×104cells/well) for 6 days and three wells per day were counted. The average cell counts at each time point were then plotted against time and the population doubling time (PDT) was determined. Then, vials of cells (2×106cells/mL) were cryopreserved for 1 month and after thawing, the cell viability was evaluated. The PDT of the second passage was about 23 h and for the eighth passage was about 30 h. The viability of the cultures was 95% in the second passage and 74.5% in the eighth passage. It was shown that the Guinea pig fetal fibroblast cell culture can be established using the adherent culture method while, after freezing, the viability indices of these cells were favorable.

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Establishment and characterization of a fibroblast cell line derived from Texel sheep

Biochemistry and Cell Biology ◽

10.1139/o09-005 ◽

2009 ◽

Vol 87 (3) ◽

pp. 485-492 ◽

Cited By ~ 11

Author(s):

Linfeng F. Li ◽

Weijun J. Guan ◽

Han Li ◽

Xueyan Z. Zhou ◽

Xiujuan J. Bai ◽

...

Keyword(s):

Cell Line ◽

Fluorescent Proteins ◽

Transfection Efficiency ◽

Chromosome Analysis ◽

Fibroblast Cell ◽

Population Doubling ◽

Established Cell Line ◽

Population Doubling Time ◽

Fibroblast Cell Line ◽

Plasmid Transfection

A Texel sheep ear marginal tissue fibroblast cell line (named TSF19) was successfully established by using a primary explant technique and cell cryoconservation technology. TSF19 cells were adherent, with a population doubling time of 24.9 h. Chromosome analysis showed that >90% of cells were diploid prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the TSF19 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, or mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pECFP-N1, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 21.8% and 46.5%. This newly established cell line will not only preserve the genetic resources of the important Texel sheep at the cell level but will also provide a valuable resource for genomic, postgenomic, somatic cloning research.

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Comparative Bioactivity Analysis for Off-the-Shelf and Culture–Rescued Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells in a Xeno- and Serum-Free Culture System

Cell Transplantation ◽

10.1177/09636897211039441 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110394

Author(s):

Minh Quang Nguyen ◽

Hue T. H. Bui ◽

Anh Nguyen Thi Tuyet ◽

Trinh Thi Hong Nhung ◽

Duc M. Hoang ◽

...

Keyword(s):

Umbilical Cord ◽

Stromal Cells ◽

Cultured Cells ◽

Cost Effective ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Serum Free ◽

Proliferation Capacity ◽

Cell Growth And Proliferation

We recently reported a standardized xeno- and serum-free culture platform to isolate and expand umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs). Comparing populations from the same passage, cells that were cryopreserved and culture-rescued exhibited characteristics similar to those of their fresh counterparts, continuously cultured cells without interim cryopreservation. The culture rescue after thawing allowed for the cells to be fully recovered. However, since it would be more cost-effective and timesaving if cryopreserved cells can be used as an off-the-shelf product, we set out to compare the bioactivity of freshly thawed UC-MSCs versus culture-rescued UC-MSCs of the same batch that were recultured for an additional passage under our xeno- and serum-free protocol. UC-MSCs showed high viability in both the freshly thawed and the re-cultured group. Both populations displayed a similar proliferation capacity which is indicated by a comparable population doubling time and colony-forming ability. Both freshly thawed and culture-rescued UC-MSCs expressed the characteristic immunophenotype and were capable of differentiating into osteocytes, chondrocytes, and adipocytes. On the other hand, culture-rescued cells appeared to be more potent in immunosuppression than freshly thawed cells. In conclusion, freshly thawed and culture-rescued cell products share comparable bioactivity in cell growth and proliferation, immunophenotype, and differentiation potential. However, the culture-rescued cells that were allowed to grow for an additional passage appear to display a more favorable immunomodulatory potential when compared to their freshly thawed parent cells.

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ID3013 Comparative characterization of murine Bone marrow mesenchymal stem cells cultured using two different supplements

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.233 ◽

2017 ◽

Vol 4 (S) ◽

pp. 25

Author(s):

Karuppiah Thilakavathy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Culture Medium ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Comparative Characterization ◽

Medium Formulation

Preclinical studies on mesenchymal stem cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. The mouse is the most widely used species for studying the characteristics of MSC. In recent years, conflicting data were reported regarding growth kinetics, surface marker profile, differentiation capacity, genetic instability or malignant transformation and so forth, that may be a result of a range of factors. One of the factors probably is the culture medium formulation. Here we have made a comparative characterization of bone marrow-derived mesenchymal stem cells (mBM-MSC), under the same experimental conditions, cultured using two common supplements, fetal bovine serum (FBS) and MesenCultTM Stimulatory Supplement (MSS). mBM-MSC isolated from the tibias of C57BL/6 mice were cultured and expanded in Dulbecco’s Modified Eagle’s Medium supplemented with either 15% FBS or 15% MSS. Clonogenic potential, population doubling time, immunophenotyping, differentiation immunosuppression potentials and chromosome analysis of early and late passage of mBM-MSC were assessed. The findings showed that the immunophenotype and differentiation potential of mBM-MSC were similar when cultured using these supplements irrespective of passages. Variations were seen in clonogenic, growth, proliferation rate and immunosuppression potential of the mBM-MSC. This study also revealed that prolonged culture will disrupt their genetic stability regardless of the supplements used. The genetically mutated mBM-MSC were also found to maintain their stemness characteristics and immunosuppression potential. In conclusion, culture medium formulation causes variations in the cultured MSC and may influence downstream investigation findings.

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Cryopreservation of Iranian Markhoz Goat Fibroblast Cells as an Endangered National Genetic Resource

10.21203/rs.3.rs-248342/v1 ◽

2021 ◽

Author(s):

Zahra Elyasi Gorji ◽

Parvaneh Farzaneh ◽

Ahmad Nasimian ◽

Meysam Ganjibakhsh ◽

Mehrnaz Izadpanah ◽

...

Keyword(s):

Cell Lines ◽

Genetic Resource ◽

Cultured Cells ◽

Local Population ◽

Capra Hircus ◽

Population Doubling ◽

Population Doubling Time ◽

Fibroblast Cells ◽

Primary Fibroblast

Abstract The sustainable use of local animals is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population growth process facing in the future. This study aimed to establish, characterize and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM media supplemented with 10 % FBS. The cultured cell lines were morphologically consistent with fibroblast cells. The population doubling time for DMEM-cultured cells were 23±0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with >95% frequency. Experimental assays for bacteria, fungi, yeast, and mycoplasma were reported negative. The efficiencies of VSV-G (pMDG) and lentiviral pCSGW vectors encoding fluorescent proteins showed an approximate value of 65%. Species identification for each sample was performed and confirmed correct goat cell banking without any miss- and cross-contamination. This study demonstrated the successful establishment of genetically stable fibroblast bank as a valuable genetic resource for endangered Iranian Markhoz goat breed.

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Black muntjac fibroblast cell culture and interspecies embryo reconstruction in vitro

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236207001647 ◽

2007 ◽

Vol 4 (2) ◽

pp. 157-162 ◽

Cited By ~ 1

Author(s):

Fang Jun-Shun ◽

Tao Yong ◽

Zhang Mei-Ling ◽

Liu Jian-Ming ◽

Han Wei ◽

...

Keyword(s):

Mammalian Species ◽

Eastern China ◽

Fibroblast Cell ◽

Population Doubling ◽

Population Doubling Time ◽

Developmental Potential ◽

Narrow Region ◽

Low Glucose ◽

Fibroblast Cell Culture

AbstractThe black muntjac (Muntiacus crinifrons) is a critically endangered mammalian species, confined to a narrow region of south-eastern China. In the present study, the homogeneous ear fibroblast cells of a black muntjac were respectively cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose), DMEM (high glucose) and RPMI-1640. The population doubling time of subcultured cells was not significantly different between the three different media, but cell growth was greater in DMEM (low glucose). The interspecies embryos were reconstructed using the fibroblasts of black muntjac as donors and enucleated goat or rabbit oocytes as recipients and their blastocyst rates were 0 and 11.5%, respectively. The results demonstrated that the two cytoplasts could support reprogramming of the ear fibroblasts of black muntjac, but the developmental potential of the reconstructed embryos was different.

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Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord

Stem Cells International ◽

10.1155/2016/3274054 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Ryan J. Emnett ◽

Aparna Kaul ◽

Aleksandar Babic ◽

Vicki Geiler ◽

Donna Regan ◽

...

Keyword(s):

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cultured Cells ◽

Good Manufacturing Practice ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Clonogenic Potential ◽

Therapeutic Doses

Recent studies have demonstrated that the umbilical cord (UC) is an excellent source of mesenchymal stromal cells (MSCs). However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP) standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H) followed by culture in human platelet lysate (PL) supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D). Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

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Morphological and functional characteristics of cell culture derived from the mouse nail unit

Cell and Organ Transplantology ◽

10.22494/cot.v5i1.69 ◽

2017 ◽

Vol 5 (1) ◽

pp. 62-66

Author(s):

O. Kalmukova ◽

A. Ustymenko ◽

T. Lutsenko ◽

P. Klymenko ◽

V. Kyryk

Keyword(s):

Cell Culture ◽

Growth Rate ◽

Doubling Time ◽

Cultured Cells ◽

Primary Cultures ◽

Population Doubling ◽

Synthetic Activity ◽

Population Doubling Time ◽

Proliferative Potential

Nail unit is a complex anatomical structure that is capable of rapid growth and regeneration throughout the life. Such significant reparative potential is associated with the presence different types of stem and progenitor cells, whose biology remains one of the fundamental issues today. Taking into account the active search for new stem cell sources for cell therapy, the view of the nail unit as a potential site for the localization of undifferentiated cells with stem potency is topical problem.Purpose. The study was conducted with an objective to establish the morphological, morphometric and proliferative characteristics of cultured cells isolated from the mouse nail unit.Materials and methods. Primary cultures of cells were obtained from tissue sampling, which included areas of the proximal nail fold, nail matrix and onychodermis of the FVB mouse nail organ. Cells were cultured in DMEM:F12 medium with 15 % fetal bovine serum during 6 passages. We determined the colony-forming activity, the population growth rate and doubling time, measured the area of cells, nuclei, and calculated the nuclear-cytoplasmic ratio. For cell morphology analysis, we used staining with Bemer’s hematoxylin and eosin, Heidenhain’s iron hematoxylin and May-Grünwald stain.Results. According to the morphological analysis in vitro the cells from mouse nail unit are heterogeneous with high synthetic activity and a low nuclear-cytoplasmic ratio – the features characteristic of the low-differentiated cells. The population doubling time of the culture was 80 ± 6.5 hours on average, the fastest growing cells were at the 4th passage (63 ± 7 hours). The specific growth rate for cell culture is low (0.01 ± 0.0007).The colony forming efficiency at the 5th passage was only 4 %. A significant number of colonies was small with large poorly proliferative cells, which may indicate a production of large numbers of transitional progenitor cells.Conclusion. The obtained cell culture from the mouse nail unit according to the analysis of their morphology, morphometry and proliferative potential is heterogeneous and requires the further development of pure culture technologies for the detailed characterization of separate subpopulations of cells.

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