Application of a Hyperelastic 3D Printed Scaffold for Mesenchymal Stem Cell-Based Fabrication of a Bizonal Tendon Enthesis-like Construct

After surgical tendon repair, the tendon-to-bone enthesis often does not regenerate, which leads to high numbers of rupture recurrences. To remedy this, tissue engineering techniques are being pursued to strengthen the interface and improve regeneration. In this study, we used hyperelastic biphasic 3D printed PLGA scaffolds with aligned pores at the tendon side and random pores at the bone side to mimic the native insertion side. In an attempt to recreate the enthesis, the scaffolds were seeded with adult human mesenchymal stem cells and then cultured in dual fluidic bioreactors, which allows the separate in-flow of tenogenic and chondrogenic differentiation media. MTS assay confirmed the ability of cells to proliferate in dual-flow bioreactors at similar levels to tissue culture plate. Hematoxylin-eosin staining confirmed a compact cell layer entrapped within newly deposited extracellular matrix attached to the scaffolds’ fibers and between the porous cavities, that increased with culture time. After 7, 14, and 21 days, samples were collected and analyzed by qRT-PCR and GAG production. Cultured constructs in dual fluidic bioreactors differentiate regionally toward a tenogenic or chondrogenic fate dependent on exposure to the corresponding differentiation medium. SOX9 gene expression was upregulated (up to 50-fold compared to control) in both compartments, with a more marked upregulation in the cartilaginous portion of the scaffold, By day 21, the cartilage matrix marker, collage II, and the tendon specific marker, tenomodulin, were found to be highly upregulated in the cartilaginous and tendinous portions of the construct, respectively. In addition, GAG production in the treated constructs (serum-free) matched that in control constructs exposed to 10% fetal bovine serum, confirming the support of functional matrix formation in this system. In summary, our findings have validated this dual fluidic system as a potential platform to form the tendon enthesis, and will be optimized in future studies to achieve the fabrication of distinctly biphasic constructs.

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Tissue Engineering of Fibroblast Constructs and Anisotropic Collagen Gels

MRS Proceedings ◽

10.1557/proc-724-n7.9 ◽

2002 ◽

Vol 724 ◽

Author(s):

Sarah Calve ◽

Ellen Arruda ◽

Robert Dennis ◽

Karl Grosh ◽

Krystyna Pasyk

Keyword(s):

Tissue Engineering ◽

Self Assembly ◽

Cell Layer ◽

Fetal Bovine Serum ◽

Self Organization ◽

Collagen Gels ◽

Confluent Cell ◽

Fetal Bovine

AbstractThe creation of an in vitro functional tendon construct will enable testing of the influence of mechanics and nutrients on the development and remodeling of tendon under known controlled stimuli which is difficult to achieve in vivo. Tendon constructs were engineered in vitrovia stress-mediated self organization of fibroblasts and ECM on a laminin coated elastomer substrate. Varying the laminin density and the amount of fetal bovine serum on the substrate affected the ability of tendon fibroblasts to form a confluent cell layer and the time to layer delamination. Understanding the factors that promote self-assembly of tendon constructs will enable their combination with already developed in vitro muscle constructs.

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227 MESENCHYMAL STEM CELL ISOLATION AND CULTURE FROM ADIPOSE TISSUE OF A DEAD DOG

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab227 ◽

2016 ◽

Vol 28 (2) ◽

pp. 245

Author(s):

S. Saini ◽

V. Sharma ◽

H. N. Malik ◽

S. K. Guha ◽

D. Malakar

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Blue Staining ◽

Marker Genes ◽

Specific Marker ◽

The Future ◽

Fetal Bovine

Isolation of cells or stem cells from clinically dead animals may serve applications such as revival of the animal through somatic cell nuclear transfer (SCNT) or cryopreservation of their cells for a long period so that cells can be used in the future. Thus, combining isolation of cells from clinically dead animals and SCNT of germplasm of elite animals could benefit research into endangered or extinct species. In the present study, we tried to isolate and culture adipose-derived mesenchymal stem cells (ADSC) from a clinically dead dog. Adipose tissues were collected surgically from the abdomen of a dead dog after 3 h and processed tissues within 10 h of death. The isolated tissues were washed in 70% ethanol for 30 s and washed 5 times in Dulbecco’s PBS supplemented with 50 µg mL–1 gentamicin. These fat tissues were minced to very small pieces and washed in DMEM by centrifugation at 800 rpm for 3 min. The tissue pellet was subjected to enzymatic digestion (collagenase 1 mg mL–1 of Dulbecco’s PBS) at 37°C in CO2 incubator for 1 h, with intermittent shaking after every 10 min. The digestive enzyme was inactivated by equal volume of DMEM/F-12 supplemented with fetal bovine serum (20%) and centrifuged at 1000 rpm for 10 min. The pellet was resuspended in DMEM/F-12 with 10% fetal bovine serum and cultured at 1 × 106 cells mL–1 in 25-cm2 tissue culture flasks. The medium was changed after every 48 h. Mesenchymal stem cells (MSC) were observed under an inverted microscope after 6 days. These cells were subcultured and a confluent monolayer was obtained. We have already standardized the protocol of MSC culture and characterisation as we are treating wounded and paralysed dogs using these MSC in a pet clinic. Characterisation of MSC was performed with specific surface marker genes of CD44, CD29, and CD166 in PCR and by immunocytochemistry of MSC-specific marker of CD44. Differentiation of these MSC into osteogenesis and chondrogenesis were observed after 3 weeks. Chondrogenic differentiation was confirmed by positive expression of chondrocyte-specific marker genes Aggrecan F-TTGGACTTTGGCAGAATACC and R-CTTCCACCAATGTCGTATCC and Collagen II F-AACCCTGGAACTGACGGAAT and R-CTCACCCGTTTGACCTTTCG primer in PCR. The MSC were cryopreserved after 80% confluency was reached. The monolayer cells were scraped out from the culture flask and pelleted down. The pellet was resuspended in DMEM containing 10% DMSO and 20% fetal bovine serum. The number of cells was determined by trypan blue staining using an automatic cell counter and 105 cells mL–1 were added to a 2-mL cryogenic vial. The cryogenic vials were kept in a cryobox at –80°C for slow cooling. Then these vials were transferred to liquid nitrogen tanks after 12 h for long-term storage. We conclude that ADSC were successfully cultured from adipose tissue of a dog within 10 h of death and further subcultured under in vitro conditions. The cells could be used for SCNT to revive the dead animal and cryopreserve these cells for use in the future.

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Validation of reference and identity-defining genes in human mesenchymal stem cells cultured under unrelated fetal bovine serum batches for basic science and clinical application

Stem Cell Reviews and Reports ◽

10.1007/s12015-018-9822-0 ◽

2018 ◽

Vol 14 (6) ◽

pp. 837-846 ◽

Cited By ~ 5

Author(s):

Federica Banfi ◽

Alessandra Colombini ◽

Carlotta Perucca Orfei ◽

Valentina Parazzi ◽

Enrico Ragni

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Clinical Application ◽

Bovine Serum ◽

Basic Science ◽

Fetal Bovine Serum ◽

Human Mesenchymal Stem Cells ◽

Fetal Bovine ◽

Cells Cultured

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Gradient generation by an osmotic pump and the behavior of human mesenchymal stem cells under the fetal bovine serum concentration gradient

Lab on a Chip ◽

10.1039/b710777c ◽

2007 ◽

Vol 7 (12) ◽

pp. 1673 ◽

Cited By ~ 77

Author(s):

Joong Yull Park ◽

Chang Mo Hwang ◽

Soon Hyuck Lee ◽

Sang-Hoon Lee

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Serum Concentration ◽

Concentration Gradient ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Human Mesenchymal Stem Cells ◽

Fetal Bovine Serum Concentration ◽

Fetal Bovine ◽

Gradient Generation

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Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162120 ◽

1990 ◽

Vol 48 (3) ◽

pp. 912-913

Author(s):

Jaang J. Wang ◽

Cheng C. Chen ◽

Men F. Shaio ◽

Chia T. Liu ◽

Chung S. Lee ◽

...

Keyword(s):

Culture Medium ◽

Cytopathic Effect ◽

Fetal Bovine Serum ◽

Virus Particles ◽

Electron Microscopies ◽

Embedding Technique ◽

Fetal Bovine ◽

Flat Embedding ◽

20 Nm ◽

Labeling Method

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.

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Treatment of fetal bovine serum improves early development of porcine embryos by alleviating oxidative stress

Reproduction Abstracts ◽

10.1530/repabs.1.p070 ◽

2014 ◽

Author(s):

Seon-A Choi ◽

Seong-Eun Mun ◽

Pil-Soo Jeong ◽

Hae-Jun Yang ◽

Seung-Bin Yoon ◽

...

Keyword(s):

Oxidative Stress ◽

Early Development ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Fetal Bovine

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МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

Molochnoe i miasnoe skotovodstvo ◽

10.33943/mms.2019.6.39671 ◽

2019 ◽

pp. 20-22

Author(s):

T.I. KUZMINA ◽

I.V. CHISTYAKOVA

Keyword(s):

Granulosa Cells ◽

Follicular Fluid ◽

Bovine Serum ◽

Egg Quality ◽

Fetal Bovine Serum ◽

Reproductive Technologies ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Cultural Systems ◽

Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

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