Cell Culture Period Research Articles

A direct cell printing supplemented with low-temperature processing method for obtaining highly porous three-dimensional cell-laden scaffolds.

A simple and versatile cell dispensing method for fabricating three-dimensional (3D) cell-laden alginate scaffolds using a low-temperature biofabrication process is proposed. To obtain optimum processing conditions, various low temperatures from -2 °C to -40 °C were used. Taking the pore structure (strut size, pore size and pore connectivity) and in situ cell viability into consideration, -10 °C was found to be the most appropriate processing temperature. In addition, the rapid temperature change during processing was a critically important parameter affecting initial cell viability. To examine this effect, we used various cell stabilization times during low-temperature processing. Inclusion of a stabilization time of over 60 min significantly improved cell viability compared to times below 30 min. Based on these optimized conditions (-10 °C and 60 min), we successfully fabricated various precisely controlled 3D cell-laden pore structures, and the in situ cell viabilities for osteoblast-like cell (MG63) and mesenchymal stem cell (MSC)-laden scaffolds were 84% and 70%, respectively. During long cell culture periods (20, 45 and 60 days), the cells in the alginate scaffolds survived, proliferated and even mineralized well. These results suggest that this new biofabrication process has great potential as a method for obtaining cell-laden scaffolds.

Differences in the Induction of Induced Human CD4+ CD25+ FoxP3+ T-Regulatory Cells and CD3+ CD8+ CD28− T-Suppressor Cells Subset Phenotypes In Vitro: Comparison of Phorbol 12-Myristate 13-Acetate/Ionomycin and Phytohemagglutinin Stimulation

CD4+ CD25+ FoxP3+ T-regulatory cells (Treg) and CD3+ CD8+ CD28− T-suppressor cells (Ts) were shown to have immunosuppressive function in vivo and in vitro. However, the in vitro inducibility of Ts subsets is rather unclear. We investigated the induction of Treg and Ts subsets in peripheral blood mononuclear cells of 5 healthy control individuals during stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin or phytohemagglutinin (PHA). Phenotypes were analyzed 0, 4, 8, 16, and 24 hours after initiation of cell culture using 4-color fluorescence flow-cytometry. Number of CD4+ CD25+ FoxP3+ CD127− Treg increased during PMA/ionomycin or PHA stimulation (P < .01). CD4+ CD25+ FoxP3+ Treg coexpressed the phenotypes interleukin (IL)-2−, IL-10+, and/or transforming growth factor (TGF)-β+ after stimulation (all P < .01). Interferon (IFN)-γ production was induced only by PMA/ionomycin (P < .01) but not by PHA (P = NS). IFN-γ-secreting Treg were detectable at 4 hours whereas IL-2−, IL-10+ and/or TGF-β+ Treg required 16 hours of stimulation. In contrast, CD3+ CD8+ CD28− Ts phenotypes were not inducible during 24-hour PMA/ionomycin or PHA stimulation (all P = NS). However, Ts coexpressed IL-10 and/or TGF-β during polyclonal stimulation (all P < .01), whereas the proportion of IL-2− Ts remained stable during the cell culture period (P = NS). Similar to Treg, IFN-γ-secreting Ts were detected only during PMA/ionomycin stimulation (P < .01), but not during PHA stimulation (P = NS). We conclude that the proportion of CD3+ CD8+ CD28− Ts remains stable during polyclonal stimulation. They modify only the cytokine pattern indicating activation of the Ts. In contrast, CD4+ CD25+ FoxP3+ CD127− Treg are inducible by PMA/ionomycin and PHA stimulation. IFN-γ- secreting Treg form the first line of immunoregulatory T cells during an initiated immune response followed by IL-2−, IL-10+, and/or TGF-β+ Treg.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Gene expression of MC3T3-E1 osteoblastic cells on titanium and zirconia surface

PURPOSEThis study was performed to define attachment and growth behavior of osteoblast-like cells and evaluate the gene expression on zirconia compared to titanium.MATERIALS AND METHODSMC3T3-E1 cells were cultured on (1) titanium and (2) zirconia discs. The tetrazolium-based colorimetric assay (MTT test) was used for examining the attachment of cells. Cellular morphology was examined by scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation rate. Mann-Whitney test was used to assess the significance level of the differences between the experimental groups. cDNA microarray was used for comparing the 20215 gene expressions on titanium and zirconia.RESULTSFrom the MTT assay, there was no significant difference between titanium and zirconia (P>.05). From the SEM image, after 4 hours of culture, cells on both discs were triangular or elongated in shape with formation of filopodia. After 24 hours of culture, cells on both discs were more flattened and well spread compared to 4 hours of culture. From the ALP activity assay, the optical density of E1 cells on titanium was slightly higher than that of E1 cells on zirconia but there was no significant difference (P>.05). Most of the genes related to cell adhesion showed similar expression level between titanium and zirconia.CONCLUSIONZirconia showed comparable biological responses of osteoblast-like cells to titanium for a short time during cell culture period. Most of the genes related to cell adhesion and signal showed similar expression level between titanium and zirconia.

The role of Hsp90α in heat-induced apoptosis and cell damage in primary myocardial cell cultures of neonatal rats

To understand the mechanism underlying the sudden animal death caused by acute heart failure during heat stress, the relationships among the heat-induced pathological changes and apoptosis and the variations in the levels of protective Hsp90α and its mRNA in the heat-stressed primary myocardial cells of neonatal rats in vitro were studied by cytopathological observation, immunoblotting, RT-PCR, and analysis of the related enzymes. After a period of adaptive cell culture, the myocardial cells were immediately exposed to heat stress at 42°C for 10, 20, 40, 60, 120, 240, 360, and 480 min. Levels of creatine kinase increased from the beginning of heat stress, and the cells exposed to heat stress showed acute cellular lesions characterized by vacuolar degeneration and necrosis after 40 min of heat stress, suggesting that the myocardial cells in vitro were obviously stressed and damaged by higher temperature. The levels of cleaved caspase-3 and cytochrome C, which were related to apoptosis, increased significantly after 40 min of heat stress while the Hsp90α protein level significantly decreased. In contrast, after 6 h of exposure to heat stress, the levels of cleaved caspase-3 and cytochrome C decreased while those of Hsp90α significantly increased, suggesting that early depletion of Hsp90α coincides with a high rate of necrosis and apoptosis in heat-stressed myocardial cells, while the Hsp90α level in surviving cells increases again with significantly less apoptosis after 6 h of heat stress. These findings also indicate that apoptosis of myocardial cells occurs through the activation of the cytochrome C and caspase-3 pathway. The cell repair capacity of Hsp90α is overstrained in the early phase of heat treatment and needs some hours to stabilize. As a result, in the primary myocardial cells in vitro, Hsp90α shows protective activity against damage at the end period of the heat exposure.

Three-Dimensional Culture of Vascular Endothelial Cells Using Vascular Endothelial Growth Factor-Loaded Apatite-Fiber Scaffolds with Enhanced Mechanical Property

We have successfully developed the apatite-fiber scaffold (AFS) with enhanced mechanical porosity for tissue engineering of bone and liver via two routes: i) use of two type of carbon beads with diameter of ~150 μm and ~20 μm and following ii) uniaxial pressing of the green compacts. Our Aim is to add vascular formation ability into the above AFS in order to maintain the regenerated tissues for a long time. In the present study, the AFSs with various porosities (68±2.4, 85±1.5, 89±0.6, 92±1.0%) were fabricated, and then loaded with vascular endothelial growth factor (VEGF). Drug release from VEGF-loaded AFSs with various porosities was examined by immersing them into phosphate buffer. The AFSs with the highest porosity (92%) could be released with the most VEGF among examined AFSs. In addition, we carried out preliminary study for the compatibility of vascular endothelial cells, M1 cells established by Matsuura et al. to the VEGF-loaded AFS (porosity: 92%), in order to account for the vascular formation into the pore of the AFS. The numbers of M1 cells cultured in/on the VEGF-loaded AFS were about 1.5 times that of VEGF-free AFS over a period of cell culture. These results demonstrate that the VEGF-loaded AFS with enhanced mechanical property have a good compatibility to the M1 cells as a model of vascular endothelial cells.

Scalable production of controllable dermal papilla spheroids on PVA surfaces and the effects of spheroid size on hair follicle regeneration

Organ size and numbers are vital issues in bioengineering for hair follicle (HF) regeneration. Murine HF dermal papilla (DP) cells are able to induce HF neogenesis when transplanted as aggregates. However, how the preparation of murine and human DP aggregates affects HF inductivity and the size of regenerated HF is yet to be determined. Here we report a scalable method for production of controllable human and rat DP spheroids in general labs for reproducible experiments. Compared with more hydrophobic polyethylene and poly(ethylene-co-vinyl alcohol), DP cells are poorly adhesive to hydrophilic polyvinyl alcohol (PVA). Seeded in PVA-coated 96-welled commercial PCR tube arrays, DP cells quickly aggregate into single spheroids with progressive compaction. Varying seeded cell numbers and culture periods enables us to control the size and cell number of the spheroids. The spheroids obtained have high viability and preserve DP characters. A proof of principle experiment was conducted to examine the size effect on the efficiency and efficacy of HF regeneration. We found that both human and rat DP spheroids are able to induce HF neogenesis and larger DP spheroids exhibit higher HF inductivity. However, the average diameter of regenerated hair fiber did not significantly change with the increasing size of transplanted DP spheroids. The result suggests that an appropriate size of DP spheroid is essential for HF inductivity, but its size cannot be directly translated to a thicker regenerated hair. Our results also have implications on the efficiency and efficacy in the regeneration of other epithelial organs.

Pushing the Reset Button: Chemical-Induced Conversion of Amniotic Fluid Stem Cells Into a Pluripotent State

In an article by Moschidou et al. appearing in this issue of Molecular Therapy, a major step toward factor-free derivation of a pluripotent cell type was made using a c-Kit+ subpopulation of human first-trimester amniotic fluid cells, which could be converted into bona fide induced pluripotent stem (iPS) cells without the ectopic expression of the pluripotency factors.1 After an extended cell culture period and exposure to the histone deacetylase (HDAC) inhibitor valproic acid (VPA), these so-called amniotic fluid stem cells (AFSCs) could be reset into a pluripotent state.

Cells (MC3T3-E1)-Laden Alginate Scaffolds Fabricated by a Modified Solid-Freeform Fabrication Process Supplemented with an Aerosol Spraying

In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl(2)), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 μm) and an aerosol spray flow rate (0.93 ± 0.12 mL min(-1)), 10 mm s(-1) nozzle moving speed, a 10 wt % concentration of CaCl(2) in the aerosol solution, and 2 wt % concentration of CaCl(2) in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, pore-structured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm(3) and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well.

Characterization of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Bioenergetics and Utilization in Safety Screening

Cardiotoxicity remains the number one reason for drug withdrawal from the market, and Food and Drug Administration issued black box warnings, thus demonstrating the need for more predictive preclinical safety screening, especially early in the drug discovery process when much chemical substrate is available. Whereas human-ether-a-go-go related gene screening has become routine to mitigate proarrhythmic risk, the development of in vitro assays predicting additional on- and off-target biochemical toxicities will benefit from cellular models exhibiting true cardiomyocyte characteristics such as native tissue-like mitochondrial activity. Human stem cell-derived tissue cells may provide such a model. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicity-profiling techniques to characterize mitochondrial function in induced pluripotent stem cell (iPSC) derived human cardiomyocytes in the presence of differing carbon sources over extended periods in cell culture. Functional analyses demonstrate that iPSC-derived cardiomyocytes are (1) capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate and (2) bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids. We utilized this model to test a variety of kinase inhibitors with known clinical cardiac liabilities for their potential toxicity toward these cells. We found that the kinase inhibitors showed a dose-dependent toxicity to iPSC cardiomyocytes grown in galactose and that oxygen consumption rates were significantly more affected than adenosine triphosphate production. Sorafenib was found to have the most effect, followed by sunitinib, dasatinib, imatinib, lapatinib, and nioltinib.

Biodentine Induces Immortalized Murine Pulp Cell Differentiation into Odontoblast-like Cells and Stimulates Biomineralization

IntroductionBiodentine (Septodont, Saint Maur des Faussés, France), a new tricalcium silicate-based cement, has recently been commercialized and advertised as a bioactive material. Its clinical application and physical properties have been widely described, but, so far, its bioactivity and biological effect on pulp cells have not been clearly shown. Thus, the aim of this study was to evaluate the biological effect of Biodentine on immortalized murine pulp cells (OD-21). MethodsOD-21 cells were cultured with or without Biodentine. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay after 2, 3, and 5 days of stimulation. The expression of several biomolecular markers was analyzed to screen differentiation pathways, both on a gene level with Real-time reverse transcription polymerase chain reaction and on a protein level by measuring alkaline phosphatase activity. Alizarin red staining was used to assess and quantify biomineralization. ResultsThe expression patterns of several genes confirmed the differentiation of OD-21 cells into odontoblasts during the period of cell culture. Our results suggest that Biodentine is bioactive because it increased OD-21 cell proliferation and biomineralization in comparison with controls. ConclusionsBecause of its bioactivity, Biodentine can be considered as a suitable material for clinical indications of dentin-pulp complex regeneration, such as direct pulp capping.

Precut Verfahren für Descemet-Membran-Endothelzelltransplantation, Präparation und Aufbewahrung in Kultur

The preparation of the Descemet's membrane (DM) with the endothelial cell layer may be performed directly prior to surgery or as a precut tissue procedure. The purpose of the current study was the evaluation of the preparation technique and the tissue culture of 10 days regarding potential endothelial cell loss. Ten corneoscleral rims with an average age of 64.3 years were dissected to obtain 8.5 mm in diameter endothelial-DM complexes, which subsequently were organ cultured for 10 days. The endothelial cell density (ECD) was assessed during the cell culture period at days 1., 4., 7. and 10. In addition, time of preparation and transplant morphology were evaluated. The DM with the endothelial cell layer could successfully be dissected from all corneoscleral rims. The average preparation time was 8.3 min. The average ECD count was 2183 ± 77 cells/mm2 prior to, 2094 ± 110 cells/ mm2 at day 1, 2078 ± 134 cells/mm2 at day 4, 1977 ± 107 cells/mm2 at day 7 and 1898 ± 170 cells/mm2 at day 10 after preparation, respectively. Endothelial cell loss was 4.1 %, 4.8 %, 9.4 % and 13.1 % after preparation, respectively. None of the transplants exhibited large, centrally-located cell deficits. The isolated storage of DM with the endothelial layer, without any stromal remnants, showed gratifying results under storage conditions in organ culture with a moderate ECD decrease. Hence, the implementation of a precut DMEK is conceivable.

Factorial Study of Compressive Mechanical Properties and Primary In Vitro Osteoblast Response of PHBV/PLLA Scaffolds

For bone tissue regeneration, composite scaffolds containing biodegradable polymers and nanosized osteoconductive bioceramics have been regarded as promising biomimetic systems. Polymer blends of poly(hydroxybutyrate‐co‐hydroxyvalerate) (PHBV) and poly(L‐lactic acid) (PLLA) can be used as the polymer matrix to control the degradation rate. In order to render the scaffolds osteoconductive, nano‐sized hydroxyapatite (nHA) particles can be incorporated into the polymer matrix. In the first part of this study, a factorial design approach to investigate the influence of materials on the initial compressive mechanical properties of the scaffolds was studied. In the second part, the protein adsorption behavior and the attachment and morphology of osteoblast‐like cells (Saos‐2) of the scaffolds in vitro were also studied. It was observed that nHA incorporated PHBV/PLLA composite scaffolds adsorbed more bovine serum albumin (BSA) protein than PHBV or PHBV/PLLA scaffolds. In vitro studies also revealed that the attachment of human osteoblastic cells (SaOS‐2) was significantly higher in nHA incorporated PHBV/PLLA composite scaffolds. From the SEM micrographs of nHA incorporated PHBV/PLLA composite scaffolds seeded with SaOS‐2 cells after a 7‐day cell culture period, it was observed that the cells were well expanded and spread in all directions on the scaffolds.

Tissue Engineering for the Neonatal and Pediatric Patients

Of all the surgical specialties, the remit of the pediatric surgeon encompasses the widest range of organ systems and includes disorders from the fetus to the adolescent. As such, the recent emergence of tissue engineering is of particular interest to the pediatric surgical community. The individual challenges of tissue engineering depend largely on the nature and function of the target tissue. In general, the main issues currently under investigation include the sourcing of an appropriate cell source, design of biomaterials for guided tissue growth, provision of a biomolecular stimulus to enhance cellular functions and the development of bioreactors to allow for prolonged periods of cell culture under specific physiological conditions. This review aims to provide a general overview of tissue engineering in the major organ systems, including the cardiovascular, digestive, urinary, respiratory, musculoskeletal, nervous, integumentary and lymphatic systems. Special attention is paid to pediatrics as well as recent clinical applications.

Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

MiR-125b Regulates Calcification of Vascular Smooth Muscle Cells

Vascular calcification is a prominent feature of atherosclerosis and is closely linked to osteoporosis. Cellular differentiation is regulated by various microRNAs (miRs), including miR-125b, which is known to be involved in osteoblast differentiation. However, no specific miR has been defined that modulates vascular calcification. Herein, we assessed the impact of miR-125b in osteogenic transformation of vascular smooth muscle cells. Osteogenic transdifferentiation of human coronary artery smooth muscle cells was induced by osteogenic medium and enhanced the formation of mineralized matrix, resulting in a significantly higher mineral deposition after 21 days. Increased expression of miR-125b was time-dependent in human coronary artery smooth muscle cells and diminished during osteogenic transdifferentiation. At day 21, miR-125b was significantly reduced (-42%) compared with that in the untreated control. The expression of miR-processing enzymes, RNase III endonucleases DICER1 and DROSHA, was also decreased. Furthermore, inhibition of endogenous miR-125b promoted osteogenic transdifferentiation, as measured by increased alkaline phosphatase activity and matrix mineralization. Expression analysis revealed the osteoblast transcription factor SP7 (osterix) as a target of miR-125b. In vivo, miR-125b was decreased in calcified aortas of apolipoprotein E knockout mice. In conclusion, our results suggest that miR-125b is involved in vascular calcification in vitro and in vivo, at least partially by targeting SP7. Evaluating the role of miRs in arterial calcification in vivo may have important therapeutic implications.

Cell-Based Fabrication of Organic/Inorganic Composite Gel Material.

Biomaterials containing components similar to the native biological tissue would have benefits as an implantable scaffold material. To obtain such biomimetic materials, cells may be great contributors because of their crucial roles in synthetic organics. In addition, the synthesized organics—especially those derived from osteogenic differentiated cells—become a place where mineral crystals nucleate and grow even in vitro. Therefore, to fabricate an organic/inorganic composite material, which is similar to the biological osteoid tissue, bone marrow derived mesenchymal stem cells (BMSCs) were cultured in a 3D fibrin gel in this study. BMSCs secreted bone-related proteins that enhanced the biomineralization within the gel when the cells were cultured with an osteogenic differentiation medium. The compositions of both synthesized matrices and precipitated minerals in the obtained materials altered depending on the cell culture period. The mineral obtained in the 3D gel showed low crystalline hydroxyapatite. The composite materials also showed excellent osteoconductivity with new bone formation when implanted in mice tibiae. Thus, we demonstrated the contributions of cells for fabricating implantable organic/inorganic composite gel materials and a method for controlling the material composition in the gel. This cell-based material fabrication method would be a novel method to fabricate organic/inorganic composite biomimetic materials for bone tissue engineering.

The interactions of astrocytes and fibroblasts with defined pore structures in static and perfusion cultures

Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.

Influence of zinc and zinc chelator on HT-29 colorectal cell line

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

In vitro studies on the growth and proliferation characteristics of rat bone mesenchymal stem cells

This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease. Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates. Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day. The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.