D,L-polylactide degradation in biological media: experiment and model

2020 ◽  
pp. 40-51
Author(s):  
I. V. Arutyunyan ◽  
◽  
P. I. Borovikov ◽  
A. G. Dunaev ◽  
L. I. Krotova ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Stem Cells ◽  
Culture Medium ◽  
Molecular Mass Distribution ◽  
Hydrolytic Degradation ◽  
Living Cells ◽  
Short Length ◽  
Biological Media ◽  
Polymer Sample ◽  
Metabolic Products

The processes of changes in the surface morphology and internal structure, as well as the molecular mass distribution of amorphous D, L polylactides during their hydrolytic degradation in the presence of extra-germinal mesenchymal stem cells (MSCs) (ratschwart jelly umbilical cord) of the rat and their metabolic products have been studied. It was shown that the degradation of initially monolithic polymer samples in culture and conditioned media occurs almost identically. However, in a culture medium containing MSCs, this process is much more intense. This effect can be interpreted in terms of the influence of enzymes secreted by living cells, which diffuse from the surface into the polymer sample and accelerate its hydrolysis, entering into a catalytic reaction with the ether bonds of polylactide molecules. A mathematical model has been developed and verified that takes into account both non-catalytic and catalytic channels of hydrolysis, changes in the porosity of the polymer sample, diffusion of short-length oligomers, and adequately interprets the experimental results.

3H-Thymidine Influence on DNA Double Strand Breaks Induction in Cultured Human Mesenchymal Stem Cells

2018 ◽  
Vol 63 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Н. Воробьева ◽  
N. Vorob'eva ◽  
В. Уйба ◽  
V. Uyba ◽  
О. Кочетков ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Medium ◽  
Specific Radioactivity ◽  
Human Mesenchymal Stem Cells ◽  
Living Cells ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Immunocytochemical Analysis ◽  
Γh2ax Foci

Purpose: To estimate the impact of 3H-thymidine on DNA double strand breaks (DSBs) induction in cultured human mesenchymal stem cells (MSC). Material and methods: Isolation and cultivation of human bone marrow MSC was carried out according to a standard procedure. A sterile solution of 3H-thymidine with different specific radioactivity was added to the cell culture and incubated under the conditions of the CO2 incubator for 24 hours. The specific radioactivity of 3H-thymidine in the incubation medium was 50–1600 kBq/ml. To evaluate quantitatively the DSBs, an immunocytochemical analysis of the DSB marker – γH2AX foci histone was used. Additionally, the proportion of dividing cells was estimated using an immunocytochemical analysis of the cell proliferation marker, the Ki67 protein. Results: It was shown that 24 h incubation of human MSC in a culture medium results in a dose-dependent increase in γH2AX foci. There is a linear increase in the foci γH2AX in the range of 50–400 kBq/ml, after which the relative quantitative yield of foci per unit of specific radioactivity begins to decrease. In general, the dose-effect relationship is approximated by the quadratic function y = 3.13 + 50.80x – 12.38x2 (R2 = 0.99), where y is the number of foci γH2AX in the cell nucleus, and x is the specific radioactivity in 1000 kBq/ml. It was found that incubation of human MSC in a culture medium containing 800 and 1600 kBq/ml of 3H-thymidine resulted in a statistically significant decrease in the cells proliferative activity compared to the control of ~1.25 and 1.41 respectively. The peculiar biological limitation of tritium accumulation in the cell nucleus explains well the nonlinear character of the dependence of the formation of DSBs on the specific radioactivity of 3H-thymidine in the culture medium observed in our study. Conclusion: Quantitative analysis of γH2AX foci has proved to be a highly reproducible and highly sensitive method for evaluating the induction of DSBs in living cells under the action of 3H-thymidine. An analysis of the foci of γH2AX will be useful for accurate estimating the quantitative yield of DBS in living cells per dose of 3H-thymidine β-radiation. To do this, it is necessary to make a correct calculation of the doses received by the cells taking into account the microdistribution of 3H-thymidine in the cell volume and its accumulation in the DNA of living cells.

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

Nitrification: an old process, a new concern

1971 ◽  
Vol 24 ◽  
Author(s):  
W. H. Verstraete
Keyword(s):  
Culture Medium ◽  
Nitrogen Sources ◽  
Living Cells ◽  
Factors Affecting ◽  
Inhibiting Effect ◽  
Proteose Peptone ◽  
Asparaginase Activity

Some  factors affecting the L-asparaginase activity of E.  aroideae were investigated. Increasing  concentrations of glucose in the culture medium had an inhibiting effect on  the production of L-asparaginase by this microorganism. Buffering of the  culture medium in order to stabilize the pH during growth resulted in a decrease  of the L-asparaginase activity. From the different nitrogen sources examined,  tryptone, proteose peptone nr 2 and nr 3 stimulated the L-asparaginase  production. Toluene treatment of the cells practically destroyed the  L-asparaginase. Acetone dried cells showed an L-asparaginase activity  comparable with the activity of living cells.

Toward a predictive theoretical model for osmolality rise with non-humidified incubation: a randomized, multivariate response-surface study

2021 ◽  
Author(s):  
Steven F Mullen
Keyword(s):  
Mathematical Model ◽  
Response Surface ◽  
Surface Area ◽  
Embryo Culture ◽  
Culture Medium ◽  
Conflicts Of Interest ◽  
Volume Ratio ◽  
P Value ◽  
Cook Medical ◽  
Over Time

Abstract STUDY QUESTION What factors associated with embryo culture techniques contribute to the rate of medium osmolality change over time in an embryo culture incubator without added humidity? SUMMARY ANSWER The surface area-to-volume ratio of culture medium (surface area of the medium exposed to an oil overlay), as well as the density and height of the overlaying oil, all interact in a quantitative way to affect the osmolality rise over time. WHAT IS KNOWN ALREADY Factors such as medium volume, different oil types, and associated properties, individually, can affect osmolality change during non-humidified incubation. STUDY DESIGN, SIZE, DURATION Several experimental designs were used, including simple single-factor completely randomized designs, as well as a multi-factor response surface design. Randomization was performed at one or more levels for each experiment. Osmolality measurements were performed over 7 days, with up to 8 independent osmolality measurements performed per treatment group over that time. For the multi-factor study, 107 independent combinations of factor levels were assessed to develop the mathematical model. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was conducted in a research laboratory setting. Commercially available embryo culture medium and oil was used. A MINC incubator without water for humidification was used for the incubation. Osmolality was measured with a vapor pressure osmometer after calibration. Viscometry and density were conducted using a rheometer, and volumetric flasks with an analytical balance, respectively. Data analyses were conducted with several commercially available software programs. MAIN RESULTS AND THE ROLE OF CHANCE Preliminary experiments showed that the surface area-to-volume ratio of the culture medium, oil density, and oil thickness above the medium all contributed significantly (P < 0.05) to the rise in osmolality. A multi-factor experiment showed that a combination of these variables, in the form of a truncated cubic polynomial, was able to predict the rise in osmolality, with these three variables interacting in the model (P < 0.05). Repeatability, as measured by the response of identical treatments performed independently, was high, with osmolality values being ± 2 of the average in most instances. In the final mathematical model, the terms of the equation were significant predictors of the outcome, with all P-values being significant, and only one P-value > 0.0001. LIMITATIONS, REASONS FOR CAUTION Although the range of values for the variables were selected to encompass values that are expected to be encountered in usual embryo culture conditions, variables outside of the range used may not result in accurate model predictions. Although the use of a single incubator type and medium type is not expected to affect the conclusions, that remains an uncertainty. WIDER IMPLICATIONS OF THE FINDINGS Using this predictive model will help to determine if one should be cautious in using a specific system and will provide guidance on how a system may be modified to provide improved stability during embryo culture. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Cook Medical. The author is a Team Lead and Senior Scientist at Cook Medical. The author has no other conflicts of interest to declare TRIAL REGISTRATION NUMBER N/A.

scholarly journals High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

2019 ◽  
Vol 6 (6) ◽  
pp. 3213-3221
Author(s):  
Hieu Liem Pham ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Medium ◽  
Diabetic Patients ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.  

scholarly journals A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells

2018 ◽  
Vol 14 (9) ◽  
pp. 421 ◽  
Author(s):  
Wan-Long Tan ◽  
Yong-Tong Zhu ◽  
Chun-Yan Wang ◽  
Shi-Yu Pang ◽  
Cheng-Yong Lei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Culture Medium ◽  
Serum Free ◽  
Differential Adhesion ◽  
Modified Method

scholarly journals Glass beads load macromolecules into living cells

1987 ◽  
Vol 88 (5) ◽  
pp. 669-678
Author(s):  
P.L. McNeil ◽  
E. Warder
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Cultured Cells ◽  
Cell Monolayer ◽  
Cell Loss ◽  
Living Cells ◽  
Glass Beads ◽  
Cell Type ◽  
Large Numbers ◽  
Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

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