scholarly journals An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

2021 ◽  
Author(s):  
Shojiro Katoh ◽  
Hiroshi Yoshioka ◽  
Shoji Suzuki ◽  
Hiroyuki Nakajima ◽  
Masaru Iwasaki ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Hyaline Cartilage ◽  
Three Dimensional ◽  
Cartilage Tissue ◽  
Human Cartilage ◽  
Tissue Samples ◽  
Chondrocyte Implantation ◽  
Initial Processing ◽  
Regenerative Therapies

Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco Modified Eagle Medium (DMEM) are commonly employed at 4-8 deg C. In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (~25 deg C) and ii) ECS transport at 4 deg C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

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

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.

scholarly journals Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuzhao Huang ◽  
Yuchen He ◽  
Meagan J. Makarcyzk ◽  
Hang Lin
Keyword(s):  
Untreated Control ◽  
Cell Number ◽  
Cartilage Tissue ◽  
Population Doubling ◽  
Control Group ◽  
Chondrocyte Implantation ◽  
Cartilage Injuries ◽  
Pre Treatment ◽  
Articular Cartilage Injuries

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.

scholarly journals iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sun Young Lee ◽  
Sung Bum Park ◽  
Young Eun Kim ◽  
Hee Min Yoo ◽  
Jongki Hong ◽  
...  
Keyword(s):  
Metabolic Disorders ◽  
Cultured Cells ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Proteomic Profiling ◽  
Esi Ms ◽  
Mitochondrial Fatty Acid ◽  
2D And 3D

AbstractThe demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

scholarly journals Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: An in vitro study

2006 ◽  
Vol 56 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Nathalie W. D. Jansen ◽  
Goris Roosendaal ◽  
Johannes W. J. Bijlsma ◽  
Jeroen DeGroot ◽  
Floris P. J. G. Lafeber
Keyword(s):  
Cartilage Damage ◽  
In Vitro Study ◽  
Vitro Study ◽  
Cartilage Tissue ◽  
Human Cartilage ◽  
Low Concentrations ◽  
Short Period

Redifferentiated Chondrocytes in Fibrin Gel for the Repair of Articular Cartilage Lesions

2019 ◽  
Vol 47 (10) ◽  
pp. 2348-2359 ◽  
Author(s):  
Vanessa J. Bianchi ◽  
Adrienne Lee ◽  
Jesse Anderson ◽  
Justin Parreno ◽  
John Theodoropoulos ◽  
...  
Keyword(s):  
Hyaline Cartilage ◽  
Critical Size ◽  
Cartilage Tissue ◽  
Cell Phenotype ◽  
Fibrin Gel ◽  
High Density Culture ◽  
Defect Repair ◽  
Fibrin Clots

Background: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor β (TGFβ) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site. Purpose: To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects. Study Design: Controlled laboratory study. Methods: Rabbit and human chondrocytes were serially passaged twice in monolayer culture. Twice-passaged cells were used directly (dedifferentiated) or redifferentiated in high-density culture with TGFβ3. Dedifferentiated or redifferentiated cells were mixed with fibrin gel to form fibrin clots, which were cultured in vitro to assess the use of fibrin gel as a scaffold or implanted in vivo in a critical-size osteochondral defect in New Zealand White rabbit knee joints. Rabbits were sacrificed 6 weeks after implantation, and tissues were assessed histologically and by immunohistochemistry. Results: Redifferentiation of passaged chondrocytes by means of 3-dimensional culture in the presence of TGFβ3 improved the formation of cartilaginous tissues in vitro, and culture in fibrin gel did not affect the cell phenotype. Implantation of dedifferentiated cells in vivo resulted in fibrocartilaginous repair tissues. Redifferentiated chondrocyte implants resulted in granulation tissues containing the hyaline cartilage marker collagen type 2. Conclusion: Redifferentiated chondrocytes will maintain their chondrogenic differentiation in fibrin clots. Implanted redifferentiated chondrocytes show a different reparative response than dedifferentiated chondrocytes and do not appear to enhance repair at an early time point. Another study of longer duration is required to assess tissue maturation over time. Clinical Relevance: Redifferentiation of passaged chondrocytes with TGFβ3 before implantation does not improve defect repair in the first 6 weeks.

scholarly journals Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

2018 ◽  
Vol 47 (4) ◽  
pp. 1729-1741 ◽  
Author(s):  
Ronald Lützenberg ◽  
Kendrick Solano ◽  
Christoph Buken ◽  
Jayashree Sahana ◽  
Stefan Riwaldt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microarray Analysis ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Tunel Staining ◽  
Human Cartilage ◽  
Rhodamine Phalloidin ◽  
The Impact

Background/Aims: Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Methods: Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Results: Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Conclusions: Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes.

scholarly journals Novel Image Analysis Approach Quantifies Morphological Characteristics of 3D Breast Culture Acini with Varying Metastatic Potentials

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
Lindsey McKeen Polizzotti ◽  
Basak Oztan ◽  
Chris S. Bjornsson ◽  
Katherine R. Shubert ◽  
Bülent Yener ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Image Analysis ◽  
Three Dimensional ◽  
Morphological Characteristics ◽  
Tissue Samples ◽  
Metastatic Cascade ◽  
Automated Grading

Prognosis of breast cancer is primarily predicted by the histological grading of the tumor, where pathologists manually evaluate microscopic characteristics of the tissue. This labor intensive process suffers from intra- and inter-observer variations; thus, computer-aided systems that accomplish this assessment automatically are in high demand. We address this by developing an image analysis framework for the automated grading of breast cancer inin vitrothree-dimensional breast epithelial acini through the characterization of acinar structure morphology. A set of statistically significant features for the characterization of acini morphology are exploited for the automated grading of six (MCF10 series) cell line cultures mimicking three grades of breast cancer along the metastatic cascade. In addition to capturing both expected and visually differentiable changes, we quantify subtle differences that pose a challenge to assess through microscopic inspection. Our method achieves 89.0% accuracy in grading the acinar structures as nonmalignant, noninvasive carcinoma, and invasive carcinoma grades. We further demonstrate that the proposed methodology can be successfully applied for the grading ofin vivotissue samples albeit with additional constraints. These results indicate that the proposed features can be used to describe the relationship between the acini morphology and cellular function along the metastatic cascade.

scholarly journals Follicle Viability after Vitrification of Bovine Ovarian Tissue

2017 ◽  
Vol 39 (11) ◽  
pp. 614-621 ◽  
Author(s):  
Janaína Guedes ◽  
Jhenifer Rodrigues ◽  
Ana Campos ◽  
Camila Moraes ◽  
João Caetano ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Ovarian Tissue ◽  
Survival Rates ◽  
Three Dimensional ◽  
Fresh Tissue ◽  
Tissue Samples ◽  
Significant Difference ◽  
The Impact ◽  
Enzymatic Methods

Purpose The present study aimed to evaluate the impact of vitrification on the viability of follicles using a three-dimensional (3D) in vitro culture. Methods Bovine ovarian tissue samples (n = 5) obtained from slaughterhouses were utilized. The cortex was cut into small fragments of 2 × 3 × 0.5 mm using a tissue slicer. From these fragments, secondary follicles were first isolated by mechanical and enzymatic methods, then encapsulated in alginate gel and individually cultured for 20 days. Additional fragments of the same ovarian tissue were vitrified in a solution containing 25% glycerol and 25% ethylene glycol. After warming, the follicles underwent the same follicular isolation process that was performed for the fresh follicles. Results A total of 61 follicles were isolated, 51 from fresh ovarian tissue, and 10 from vitrified tissue. After the culture, the vitrified and fresh follicles showed 20% and 43.1% survival rates respectively (p = 0.290), with no significant differences. At the end of the culture, there were no significant differences in follicular diameter between the vitrified (422.93 ± 85.05 µm) and fresh (412.99 ± 102.55 µm) groups (p = 0.725). Fresh follicles showed higher mean rate of antrum formation when compared with vitrified follicles (47.1% and 20.0% respectively), but without significant difference (p = 0.167). Conclusions The follicles were able to develop, grow and form antrum in the 3D system after vitrification, despite the lower results obtained with the fresh tissue.

Sign in / Sign up

Export Citation Format

Share Document