Extended Long-Term Culture of MSC-Laden Agarose Constructs Does Not Produce Functional Tissue Comparable to Primary Chondrocytes

2010 ◽  
Author(s):  
Alice H. Huang ◽  
Robert L. Mauck
Keyword(s):  
Mechanical Properties ◽  
3D Culture ◽  
Biochemical Properties ◽  
Clinical Use ◽  
Promising Alternative ◽  
Chondrogenic Medium ◽  
Primary Chondrocytes ◽  
The Poor

Articular cartilage lines the surfaces of joints and transmits the forces arising from locomotion. The poor ability of cartilage to self-repair has motivated efforts to engineer replacements that recapitulate this load-bearing function. While chondrocyte-laden constructs have been generated with near-native mechanical properties, limitations in chondrocyte availability may preclude their clinical use. Therefore, mesenchymal stem cells (MSCs), which can undergo chondrogenesis in 3D culture, have emerged as a promising alternative [1]. However, although MSCs deposit a cartilaginous matrix, mechanical and biochemical properties are lower than those achieved with chondrocytes [1, 2]. Using microarray analysis, we recently showed that limitations in functional MSC chondrogenesis may stem from incomplete or incorrect molecular induction; molecular differences were observed between donor-matched differentiated chondrocytes and newly differentiated MSCs over 8 weeks of culture [2]. While some genes remained consistently low in MSCs compared to chondrocytes, others gradually increased with time, approaching chondrocyte levels by 8 weeks. As these molecules may underlie the functional disparity between chondrocytes and MSCs, we hypothesized that longer culture durations may improve MSC-seeded construct properties and chondrogenesis. To test this hypothesis, we characterized the evolution of functional properties of MSC- and chondrocyte-seeded constructs over 4 months of in vitro culture in pro-chondrogenic medium.

In situ generation of human brain organoids on a micropillar array

Lab on a Chip ◽  
2017 ◽  
Vol 17 (17) ◽  
pp. 2941-2950 ◽  
Author(s):  
Yujuan Zhu ◽  
Li Wang ◽  
Hao Yu ◽  
Fangchao Yin ◽  
Yaqing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
In Situ Generation ◽  
Induced Pluripotent ◽  
High Throughput Manner

We present a simple and high throughput manner to generate brain organoids in situ from human induced pluripotent stem cells on micropillar arrays and to investigate long-term brain organogenesis in 3D culture in vitro.

Leukemia Bone Marrow Primary Cells Long-Term Culture in a Biomimetic Hematopoietic Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4114-4114
Author(s):  
Li Hou ◽  
Ting Liu ◽  
Jing Tan ◽  
Wentong Meng ◽  
Li Deng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Culture System ◽  
3D Culture ◽  
Primary Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
3D Culture System ◽  
2D System

Abstract We have constructed a biomimetic hematopoietic niche (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts. Our primary results showed that the biomimetic 3D culture system is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. But so far, leukemia primary cells long-term culture from patients marrow are still difficult because it is not clear how does the regulation of leukemic cells grow ex vivo, and lack of adequate investigation between leukemic stem cells with stromal cells. Based on our previous research, we cultured bone marrow mesenchymal stem cells from chronic myelogenous leukemia (CML) patients, and conceived a “pathologic biomimetic osteoblast niche”, to explore the growth of leukemia bone marrow primary cells from CML patients. Bio-derived bone was composited with marrow mesenchymal stem cells from CML patients and constructed a 3D biomimetic osteoblast niche. The mononuclear cells (MNCs) were collected with standard Ficoll-Paque separation from newly diagnosed CML patients. The MNCs were cultured for 2∼5 weeks in the 3D culture system and compared with 2D culture system. The results showed that the proportion of CD34+ cells are increased either in 3D or 2D culture systems. Compared to input, the proportion of CD34+ cells were increased 6.52(1.87∼9)vs. 3.18(1.07∼6.8)times at 2 weeks culture, and 13.6(3.59∼26.31)vs. 7.86(0.78∼18.0)times at 5 weeks culture. The proportion of CD34+/CD38- was higher in 3D culture system than 2D system. It was 5.55(2.1∼11.7)% vs. 2.4(0.9∼3.4)%, and 13.5(3.4∼34.2)% vs. 4.83(2.1∼8.9)% at 2 weeks and 5 weeks respectively. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced more colonies than 2D system {103.33(82∼144)vs. 79(53∼122)} at 2 week culture and 47(33∼66)vs. 21.67(16∼27)at 5 week culture. LTC-IC are widely used as a surrogate in vitro culture for pluripotent stem cells, and those primitive progenitor cells responsible for leukemia in mice are named SL-IC or leukemia stem cells (LSCs). 3D system showed higher frequency of LTC-IC than that of 2D system after 2-week culture(2.23E-05(1.73∼2.56)vs.1.40E-05(1.21∼1.73)). FISH showed the proportion of Ph+ cells declined in both system during the culture, but not as rapidly as it did in 2D system{65%(3D)vs.63%(2D)at 2 week, 55%(3D)vs.35%(2D)at 5 week}, and the Ph+ cells were predominant derived from 3D culture. Our 3D culture system constructed with induced osteoblasts from mesnchymal stem cells in CML patients might provide a more suitable microenvironment for leukemic cells growing in vitro. The leukemic stem cells seemed to be regulated by the molecular signals mediated by osteoblast, and the biological characteristics of leukemia stem cells at least partially is maintained. It may be become a new method for studying leukemic HSCs/HPCs behavior in vitro.

scholarly journals Humanized Mcl-1 mice enable accurate pre-clinical evaluation of MCL-1 inhibitors destined for clinical use

2018 ◽  
Author(s):  
Margs S. Brennan ◽  
Catherine Chang ◽  
Grant Dewson ◽  
Lin Tai ◽  
Guillaume Lessene ◽  
...  
Keyword(s):  
Mouse Model ◽  
Clinical Evaluation ◽  
Transformed Cells ◽  
Clinical Use ◽  
Wild Type ◽  
Human Homologue ◽  
Cancer Models ◽  
Bh3 Mimetic

SUMMARYMCL-1 is a pro-survival BCL-2 protein required for the sustained growth of many cancers. Recently a highly specific MCL-1-inhibitor, S63845, showing 6-fold higher affinity to human compared to mouse MCL-1 has been described. To accurately test efficacy and tolerability of this BH3 mimetic drug in pre-clinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse in which MCL-1 was replaced with its human homologue. HuMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to MCL-1 inhibition. Importantly, non-transformed cells and lymphomas from huMcl-1;Eμ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eμ-Myc lymphoma cells are transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide leads to long-term remission in ~60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMCL-1 mouse model to test MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.

scholarly journals Effect of Sandblasting on the Long-Term Corrosion Resistance of Ti G4 in Artificial Saliva

2021 ◽  
Vol 6 (1) ◽  
pp. 5
Author(s):  
Patrycja Osak ◽  
Maciej Zubko ◽  
Julian Kubisztal ◽  
Joanna Maszybrocka ◽  
Bożena Łosiewicz
Keyword(s):  
Mechanical Properties ◽  
Corrosion Resistance ◽  
Machine Surface ◽  
Electrochemical Impedance ◽  
Artificial Saliva ◽  
Electron Work Function ◽  
Titanium Implants ◽  
Bone Contact

Titanium Grade 4 (G4) is the most commonly used material for dental implants due to its excellent biocompatibility and mechanical properties. However, titanium implants require a rough surface that can increase the biomechanical potential of implant–bone contact and affect protein adsorption speed. In this work, the effect of sandblasting of the Ti G4 surface on the long-term corrosion resistance in artificial saliva of pH = 7.4 at 37 °C was studied. The X-ray diffraction (XRD) single-{hkl} sin2ψ method was used to measure the sandblasted Ti residual stress. In vitro corrosion resistance tests were conducted for 21 days using the open circuit potential method, polarization curves, and electrochemical impedance spectroscopy. Using the Kelvin scanning probe, the electron work function was determined. Analysis of the obtained results showed an improvement in the corrosion resistance of the sandblasted Ti G4 compared to Ti with the machine surface. The increase in corrosion resistance was related to the residual compressive stress of 324.7 MPa present in the sandblasted Ti surface. Sandblasting caused plastic deformation of the Ti surface, which resulted in the improvement in mechanical properties, as evidenced by the increase in the hardness of the sandblasted Ti compared to Ti with the machine surface.

scholarly journals Dissociated Steroids

2007 ◽  
Vol 7 ◽  
pp. 421-430 ◽  
Author(s):  
Matthew C. Catley
Keyword(s):  
Side Effects ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Therapeutic Index ◽  
Clinical Use ◽  
Response Elements ◽  
Activation Of Transcription

Glucocorticoids (GCs) are some of the most important drugs in clinical use today. They are mainly used to suppress disease-related inflammation and are widely used for the treatment of many inflammatory diseases including asthma and arthritis. However, GCs are also associated with debilitating side effects that place limitations on the long-term use of these drugs. The development of a GC with reduced side effects would allow more effective treatments for patients who require long-term suppression of inflammation. GCs exert their effects by binding and activating the GC receptor (GR). The activated receptor then binds GC response elements (GREs) in the promoter of genes, and activates transcription (transactivation) or interferes with the activation of transcription by inhibiting the transactivating function of other transcription factors, such as AP-1 and NF-ĸB (transrepression). Transrepression is believed to be responsible for the majority of the beneficial anti-inflammatory effects of GCs, whereas transactivation is believed to play a bigger role in the unwanted side effects of GCs. Compounds that can dissociate the transactivation function of GCs from the transrepression function may, therefore, have an improved therapeutic index. A number of these dissociated corticosteroids have been developed.In vitroassays using these compounds appear to show good dissociation. However,in vivo, the dissociation appears to be lost and these compounds still produce many of the side effects associated with conventional GCs. A better understanding of the molecular mechanisms behind GC-induced effects would allow the design of novel selective GR modulators with an improved therapeutic index.

scholarly journals Polyisobutylene-Based Thermoplastic Elastomers for Manufacturing Polymeric Heart Valve Leaflets: In Vitro and In Vivo Results

2019 ◽  
Vol 9 (22) ◽  
pp. 4773 ◽  
Author(s):  
Evgeny Ovcharenko ◽  
Maria Rezvova ◽  
Pavel Nikishau ◽  
Sergei Kostjuk ◽  
Tatiana Glushkova ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Heart Valve ◽  
Heart Valves ◽  
Reference Sample ◽  
Sample Surface ◽  
Thermoplastic Elastomers ◽  
Water Contact ◽  
Promising Alternative

Superior polymers represent a promising alternative to mechanical and biological materials commonly used for manufacturing artificial heart valves. The study is aimed at assessing poly(styrene-block-isobutylene-block-styrene) (SIBS) properties and comparing them with polytetrafluoroethylene (Gore-texTM, a reference sample). Surface topography of both materials was evaluated with scanning electron microscopy and atomic force microscopy. The mechanical properties were measured under uniaxial tension. The water contact angle was estimated to evaluate hydrophilicity/hydrophobicity of the study samples. Materials’ hemocompatibility was evaluated using cell lines (Ea.hy 926), donor blood, and in vivo. SIBS possess a regular surface relief. It is hydrophobic and has lower strength as compared to Gore-texTM (3.51 MPa vs. 13.2/23.8 MPa). SIBS and Gore-texTM have similar hemocompatibility (hemolysis, adhesion, and platelet aggregation). The subcutaneous rat implantation reports that SIBS has a lower tendency towards calcification (0.39 mg/g) compared with Gore-texTM (1.29 mg/g). SIBS is a highly hemocompatible material with a promising potential for manufacturing heart valve leaflets, but its mechanical properties require further improvements. The possible options include the reinforcement with nanofillers and introductions of new chains in its structure.

Chemically- and mechanically-tunable porated polyethylene glycol gels for leukocyte integrin independent and dependent chemotaxis

TECHNOLOGY ◽  
2014 ◽  
Vol 02 (02) ◽  
pp. 133-143 ◽  
Author(s):  
Holly M. Lauridsen ◽  
Bryan J. Walker ◽  
Anjelica L. Gonzalez
Keyword(s):  
Mechanical Properties ◽  
Polyethylene Glycol ◽  
Inflammatory Diseases ◽  
New Technology ◽  
Complex Mixture ◽  
Biochemical Properties ◽  
In Vitro Models ◽  
Leukocyte Migration ◽  
Mechanical Stiffness

The extracellular matrix (ECM) is a highly complex mixture of protein, proteoglycans and growth factors that biochemically and mechanically regulates leukocyte migration during inflammation. Perturbations in ECM composition and mechanical properties are associated with the pathogenesis of chronic inflammatory diseases ranging from asthma to diabetes. The limited availability of in vitro models of human ECM has impeded inflammatory research, as current methods rely heavily on polycarbonate transwells and glass coverslips, which cannot accurately replicate the combined mechanical and biochemical properties of human ECM. Polyethylene glycol (PEG) hydrogels offer a highly tunable substrate, with respect to both mechanical properties (as a function of molecular weight) and protein conjugation; unmodified PEG, however, cannot be used for leukocyte migration studies due to its impenetrable pore networks. We present a modifi ed PEG membrane in which hydrogel pore size, pore density, mechanical stiffness, and protein presentation can be easily controlled to mimic various human ECM, providing a new technology for investigating leukocyte recruitment.

Research on the Preparation of HA/PEEK Gradient Composites by Impregnation Method and the Biosecurity

2017 ◽  
Vol 893 ◽  
pp. 35-42 ◽  
Author(s):  
Rui Ma ◽  
Ling Jun Dai ◽  
Lin Wang ◽  
Yu Dian Song
Keyword(s):  
Mechanical Properties ◽  
Bone Formation ◽  
Structural Design ◽  
Injection Moulding ◽  
In Vitro Cytotoxicity ◽  
Leach Liquor ◽  
Cytotoxicity Test ◽  
Impregnation Method ◽  
The Poor

This study prepares the HA/PEEK gradient composites with special structural design by impregnation method, which eases the contradiction of the HA/PEEK composites between the lack of bioactivity due to low HA contents and the poor mechanical properties due to high HA contents, and effectively improves the bioactivity and mechanical properties of the PEEK-based materials, and then evaluates the biosecurity of HA/PEEK gradient composites. The main functional cell of bone formation—osteoblast is adopted for the co-culture with PEEK, HA/PEEK gradient composites and the leach liquor, and the effects of PEEK and HA/PEEK gradient composites on the proliferative functions of osteoblast are studied by in vitro cytotoxicity test. The results show that no harmful admixtures are produced in the process of injection moulding, and the leach liquors of PEEK and HA/PEEK gradient composites have no effects on the proliferative functions of osteoblast, with good osteoblast compatibility.

scholarly journals Clinical Impact of Cryopreservation on Split Thickness Skin Grafts in the Porcine Model

2019 ◽  
Vol 41 (2) ◽  
pp. 306-316 ◽  
Author(s):  
Paul W Holzer ◽  
Alexandre G Lellouch ◽  
Krysta Moulton ◽  
Laurence Zhu ◽  
Zhi Yang Ng ◽  
...  
Keyword(s):  
Genetically Engineered ◽  
Cellular Viability ◽  
Skin Grafts ◽  
Porcine Skin ◽  
Clinical Value ◽  
Promising Alternative ◽  
Severe Burns ◽  
Duration Of Storage

Abstract Vital, genetically engineered, porcine xenografts represent a promising alternative to human cadaveric allografts (HCA) in the treatment of severe burns. However, their clinical value would be significantly enhanced if preservation and long-term storage—without the loss of cellular viability—were feasible. The objective of this study was to examine the direct impact of cryopreservation and the length of storage on critical in vivo and in vitro parameters, necessary for a successful, potentially equivalent substitute to HCA. In this study, vital, porcine skin grafts, continuously cryopreserved for more than 7 years were compared side-by-side to otherwise identically prepared skin grafts stored for only 15 minutes. Two major histocompatibility complex (MHC)-controlled donor–recipient pairs received surgically created deep-partial wounds and subsequent grafting with split-thickness porcine skin grafts, differentiated only by the duration of storage. Clinical and histological outcomes, as well as quantification of cellular viability via a series of 3-4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays, were assessed. No statistically significant differences were observed between skin grafts cryopreserved for 15 minutes vs 7 years. Parametric distinctions between xenografts stored for short- vs long-term durations could not be ascertained across independent clinical, histological, or in vitro evaluative methods. The results of this study validate the ability to reliably preserve, store, and retain the essential metabolic activity of porcine tissues after cryopreservation. Plentiful, safe, and readily accessible inventories of vital xenografts represent an advantageous solution to numerous limitations associated with HCA, in the treatment of severe burns.

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