Greater Cell Viability Research Articles

Biomaterials for neural-tissue engineering — Chitosan supports the survival, migration, and differentiation of adult-derived neural stem and progenitor cells

Neural precursor cells (NPCs or stem and progenitor cells) are promising in transplantation strategies to treat an injury to the central nervous system, such as a spinal cord injury (SCI), because of their ability to differentiate into neurons and glia. Transplantation studies to date have met with limited success for a number of reasons, including poor cell survival. One way to encourage cell survival in injured tissue is to provide the cells with a scaffold to enhance their survival, their integration, and potentially their differentiation into appropriate cell types. Towards this end, four amine-functionalized hydrogels were screened in vitro for adult murine NPC viability, migration, and differentiation: chitosan, poly(oligoethylene oxide dimethacrylate-co-2-amino ethyl methacrylate), blends of poly(oligoethylene oxide dimethacrylate-co-2-amino ethyl methacrylate), and poly(vinyl alcohol), and poly(glycerol dimethacrylate-co-2-amino ethyl methacrylate). The greatest cell viability was found on chitosan at all times examined, Chitosan had the greatest surface amine content and the lowest equilibrium water content, which likely contributed to the greater NPC viability observed over three weeks in culture. Only chitosan supported survival of multipotent stem cells and the differentiation of the progenitors into neurons, astrocytes, and oligodendrocytes. Plating intact NPC colonies revealed greater cell migration on chitosan relative to the other hydrogels. Importantly, long term cultures on chitosan showed no significant difference in total cell counts over time, suggesting no net cell growth. Together, these findings reveal chitosan as a promising material for the delivery of adult NPC cell-based therapies.

In Vitro Effects of Acid and Pepsin on Mouse Middle Ear Epithelial Cell Viability and MUC5B Gene Expression

To examine whether in vitro exposure of mouse middle ear epithelial cells (mMEECs) to conditions that mimic physiologic reflux upregulates Muc5b gene expression and alters cell viability. In vitro mMEEC model. Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC. Cells from the immortalized mMEEC line. Cell viability, the quantity of Muc5b messenger RNA abundance, and Muc5b promoter activity. The 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrated an acidic dose-dependent decrease in cell survival, with pH less than 4 significantly decreasing viability at 1 hour. Pepsin had a mild protective effect up to 8 hours, with greater cell viability, in the pH range of 5.0 to 7.6. Reverse-transcriptase polymerase chain reaction demonstrated induction of Muc5b messenger RNA levels over controls after exposure to acidic pH levels of 5.7, and 4 with and without pepsin. Similarly, a pH of 4.0 significantly increased Muc5b promoter activation 5.4-fold. Pepsin at neutral or acidic pH values did not significantly alter Muc5b expression or promoter activity. Despite decreasing cell viability, acidic pH drives middle ear epithelial Muc5b gene expression in vitro, which perhaps explains how laryngopharyngeal reflux can contribute to otitis media. Pepsin at neutral or acidic pH levels had minimal effects on Muc5b gene expression; thus, although pepsin may be a useful marker for detecting the presence of reflux, our results suggest that acid itself is a more likely pathologic component of gastric juice in the middle ear.

Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro

This study is to evaluate the cytotoxicity of Activ GP and RealSeal sealers in a cell culture system in vitro, and to compare them with traditional AH 26 and Kerr sealers. Samples of 0.5 mg freshly mixed or set RealSeal, Activ GP, AH 26, and Kerr sealers were eluted with 200, 400, 800, and 1,200 microL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well and cultured with 100 microL eluate from each eluate group. Cells cultured with culture medium only served as a control. After 24 hours' incubation the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with 1-way analysis of variance. For the freshly mixed sealer, cell viability in the AH 26 group was less than in all of the other 3 sealer groups. The Kerr sealer group had greater cell viability than RealSeal and Activ GP groups. For the set sealer, cell viability in the AH 26 group was greater than in all of the other 3 groups. Cell viability in the RealSeal group was less than in the Kerr and Activ GP groups. Freshly mixed RealSeal and Activ GP sealers have lower cytotoxicity than AH 26 sealer and more cytotoxicity than Kerr sealer. When sealers are set, RealSeal sealer has more cytotoxicity than AH 26 and Kerr sealer. Activ GP sealer has more cytotoxicity than AH 26 and is similar to Kerr sealer.

Biocompatibility of injectable chitosan–phospholipid implant systems

Injectable biomaterials are desirable therapeutic platforms due to minimal invasiveness and improved patient compliance, and are applicable in such areas as compound delivery and tissue engineering. The present work examined the biocompatibility of injectable blends composed of chitosan, phospholipid and lauric aldehyde (PoLi gel-LA) or lauric chloride (PoLi gel-LCl). In vitro cytotoxicity was evaluated in L929 and HeLa cell lines. Both blends resulted in acceptable biocompatibility, although greater cell viability was seen with PoLi gel-LA. In vivo biocompatibility was investigated in healthy CD-1 mice. Subcutaneous injection of the PoLi gel-LA blend caused no local or systemic toxicities over a four-week period while the PoLi gel-LCl caused immediate local toxicity. Mice injected intraperitoneally with PoLi gel-LA did not show physical or behavioural alterations, and body weight changes did not differ from control animals. Furthermore, histological examination of spleen and liver showed unaltered morphology. Interleukin-6 levels in mice injected with PoLi gel-LA did not differ from levels of control animals (6.91 ± 3.61 pg/mL versus 6.92 ± 5.02 pg/mL, respectively). Biodegradation occurred progressively, with 7.4 ± 5.02% of the original injected mass remaining after four weeks. Results obtained herein establish the biocompatibility of PoLi gel-LA and indicate its potential for use in various localized therapeutic applications.

유청단백질농축물을 기본 배지로 한 Bacillus polyfermenticus SCD균의 생육과 항산화물질 생산

The cell growth and antioxidant activity of Bacillus polyfermenticus SCD were studied in tryptic soy broth (TSB) medium and whey protein concentrate (WPC)-based medium. Overall, higher lactose contents in WPC-35 medium (up to 2.0%), and longer culture times correlated with greater cell viability. In WPC-35 medium with 1.5% and 2.0% lactose, the cell growth of B. polyfermenticus SCD was similar to growth in TSB medium. The 1,1-diphenyl-2-picyrylhydrazyl (DPPH) radical scavenging activity of culture supernatant of B. polyfermenticus SCD in WPC-35 medium was measured to assess antioxidant activity. The antioxidant activity increased up to 32 hr of culture, reaching a maximum of 75.57% DPPH radical scavenging activity. The antioxidant activity seemed to follow the typical kinetics of primary metabolite synthesis. The antioxidant activity of B. polyfermenticus SCD supernatant in WPC-35 medium was more effective and stable than supernatant from TSB medium. These results suggest that WPC-35 medium is effective for the production of antioxidant by B. polyfermenticus SCD.

Modulation of palmitic acid‐induced cell death by ergothioneine: Evidence of an anti‐inflammatory action

Inflammation and reactive oxygen species have been implicated in pathogenesis of vascular diabetic complications. However, treatment with classic free-radical scavengers and antioxidants has not been yet proved to reduce the risk of developing such complications. In search of more effective treatment we have tested the protective role of Ergothioneine (EGT), in vitro, on C2C12 cells model on FFA-induced lipotoxicity. Cells were incubated for 24 h in the presence of palmitic acid (PA) (250, 500, 750, 1000 microM), added as pro-oxidant compound, with or without 24-h pre-treatment with EGT. Cells were assessed for cell viability and MAPKs expression by Western Blot. Pre-treatment with EGT resulted in greater cell viability at each PA concentration (EGT 500 microM: 5, 16, 17, 23% and EGT 1000 microM: 9, 18, 21 and 25%). In response to PA exposure, p38 and JNK activity increased significantly while EGT prevented such activation. Moreover the analysis of the IL-6 production reveal that EGT is also able to exert anti-inflammatory action inhibiting the PA IL-6 modulation (P < 0.001). In conclusion, these results indicate that 1. EGT has a protective role on PA-induced cell death, possibly via 2. reduced activity of MAPKs cascade having also 3. an anti-inflammatory action exerted on the IL-6 modulation.

Calcium phosphate nanoparticles in biomineralization and biomaterials

Recent developments in biomineralization and biomaterials have demonstrated that nano-calcium phosphate particles play an important role in the formation of hard tissues in nature. It is suggested that the basic inorganic building blocks of bone and enamel are nanosized apatite although their hierarchical structures differ. Nanoparticles can confer on biominerals remarkable physical and chemical characteristics such as enhanced mechanical strength and self-preservation in biological fluids. In living organisms, tens to hundreds of nano-blocks, under the control of an organic matrix, combine into self-assembled biominerals. It is also confirmed experimentally that enamel- or bone-like apatite can be achieved by oriented aggregation by using nano-calcium phosphates as the raw materials, determined by the biomolecules. Interestingly, features of smaller hydroxyapatite (HAP) nanoparticles may more closely approximate features of HAP during biomineralization than features of the larger HAP particles that are conventionally used. The application and prospective use of nano-calcium phosphate in the biological repair of bone and enamel are also introduced. It is emphasized that the cell experiments reveal improved cytophilicity of nanophase calcium phosphate minerals. Greater cell viability and proliferation of bone marrow mesenchymal stem cells are measured on smaller calcium phosphates. Furthermore, studies of amorphous calcium phosphate and crystallized HAP particles are used to investigate the effect of crystallinity of nanoparticles on living cells, and show that the differentiation of stem cells is promoted significantly by nano-HAP. It is suggested that nano-HAP may be the ideal biomaterial due to its good biocompatibility and bone/enamel integration. Lots of studies of calcium phosphates are expected in the nano-zone such as for drug delivery systems and resorbable scaffolds. In this feature article, a new view on calcium phosphates highlights the importance of size and phase controls in the application of biomedical materials.

Role of hydroxyapatite nanoparticle size in bone cell proliferation

Hydroxyapatite (HAP) nanoparticles, typically 20 ± 5, 40 ± 10 and 80 ± 12 nm in diameter, were prepared and their effects on the proliferation of two bone-related cells, bone marrow mesenchymal stem cells (MSCs) and osteosarcoma cells (U2OS) were studied. The cell culture experiments showed improved cytophilicity of the nanophase mineral as compared with conventional HAP. Greater cell viability and proliferation of MSCs were measured on the nano HAP, remarkably for 20 nm sized particles. Interestingly, the growth of osteosarcoma cells was inhibited by the nano HAP and 20 nm sized particles were the best retardant. It is suggested that the HAP nanoparticles can exhibit favorable cell proliferation to optimize biological functionality, in which the particle size is believed to play a key role. These in vitro findings are of great significance for the understanding of cytophilicity and biological activity of nanoparticles during biomineralization.

Cryopreservation of cultured periosteum: Effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me 2SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at −196 °C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 °C for 30 min with Me 2SO, under agitation, showing 63.3 ± 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me 2SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me 2SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.

Controlling cell adhesion and degradation of chitosan films by N-acetylation

As part of our ongoing effort to develop a biodegradable nerve guidance channel based on chitin/chitosan, we conducted a systematic in vitro study on the biodegradation and neural cell compatibility of chitosan and N-acetylated chitosan. The in vitro degradation (pH 7.4, 37 °C) in the presence of 1.5 μg/ml lysozyme showed a progressive mass loss to greater than 50% within 4 weeks for films with 30–70% acetylation. In contrast, the degradation of samples with very low or high acetylation was minimal over the 4-week period. Neural cell compatibility of chitosan and N-acetylated chitosan was tested using primary chick dorsal root ganglion (DRG) neurons. All chitosan-based films showed DRG cell adhesion after 2 days of culture. However, cell viability decreased with increasing acetylation. Chitosan that was 0.5% acetylated had the greatest cell viability, which was approximately 8 times higher than that of chitosan that was 11% acetylated. Chitosan with 0.5% and 11% acetylation showed more and longer neurites than the other samples studied. Thus chitosan amine content can be tuned for optimal biodegradation and cell compatibility, which are important for tissue engineering in the nervous system.

The effect of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells.

Gene knockouts serve as useful experimental models to investigate the role of antioxidant enzymes in protection against oxidative stress in the lens. In the absence of gene knockout animals for Mn-containing superoxide dismutase (MnSOD), the effect of this enzyme on oxidative stress was investigated in a human lens epithelial cell line (SRA 01/04) in which the enzyme was up- or downregulated by transfection with sense and antisense expression vectors for MnSOD. Human lens epithelial cells (SRA 01/04) were transfected with plasmids containing sense and antisense human cDNA for MnSOD. The enzyme levels were determined by Western blot analysis and immunocytochemistry. The protective effect of the enzyme against the cytotoxicity of H(2)O(2) was evaluated by cell viability, DNA strand breaks, and morphologic changes observed by transmission electron microscopy. In addition, the protective effect of this enzyme against photochemically induced stress and UVB irradiation in cells was assessed by measuring the damage of cellular DNA. The MnSOD level in upregulated cells was three times higher than in downregulated cells, and the enzyme surrounded the nucleus. Cells with elevated enzyme levels were more resistant to the cytotoxic effect of H(2)O(2) with greater cell viability. MnSOD-deficient cells showed dramatic mitochondrial damage when exposed to 50 micro M H(2)O(2) for 1 hour. Similarly, oxidative challenge by H(2)O(2), photochemically generated reactive oxygen species, or UVB irradiation produced greater DNA strand breaks in MnSOD-deficient cells than in those in which the enzyme was upregulated. These findings demonstrate the protective effect of MnSOD in antioxidant defense of cultured lens epithelial cells. This approach to modulating the enzyme level in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens.

Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability

Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent. We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth). Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the expression of beta1 integrin was up-regulated only in NRKboth. Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.

Cryopreservation of Pancreatic Islets Prior to Transplantation: A Comparison between UW Solution and RPMI Culture Medium

Effective cryopreservation of pancreatic islets would be valuable in several contexts: for the assessment of islet cell viability, the measurement of β-cell function, and the maintenance of viability and sterility prior to islet transplantation. In this study, isolated rat islets were cryopreserved or not following overnight culture and the most suitable preservation solution for transportation between centers was sought. Unfrozen and frozen-and-thawed islets were allocated to each of four different groups: untreated controls; cultured overnight in RPMI at 37°C; cold stored at 4°C in RPMI for 18–24 h; and stored at 4°C in University of Wisconsin (UW) solution for 18–24 h. The greatest cell viability, as assessed by ethidium bromide/acridine orange staining and image analysis, was observed when postthawed islets were cultured in RPMI, whereas the least viable samples were those that were stored in UW solution. Measurement of insulin content and secretion in static incubation assays using 2.8 and 16.7 mM glucose showed that all treated groups exhibited a significant insulin secretory response to glucose stimulation whereas the untreated frozen–thawed islets failed to show any response. The cryopreserved islets in each group were equally successful in reversing hyperglycemia in streptozotocin-treated allogeneic rats when grafted intraportally in sufficient numbers (2000–2500). The groups also showed a similar mean graft survival time of 6–7 days before rejection. However, the best experimental group (the postthaw cultured islets) failed to cure diabetic rats when grafted in a smaller numbers (<2000). These data demonstrate prompt and sustained function in cryopreserved islets when they were maintained by any of the methods studied if they were grafted in sufficient numbers. We conclude that cold storage of thawed cryopreserved islets using either RPMI or UW solution is an effective method for their transportation and/or storage, but does not reduce their immunogenicity before transplantation.

The Membrane-bound Form of Heparin-binding Epidermal Growth Factor-like Growth Factor Promotes Survival of Cultured Renal Epithelial Cells

To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 x 10(3) cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.

Bcl-2 and Bcl-XL Can Differentially Block Chemotherapy-Induced Cell Death

Bcl-2 and its homologue Bcl-XL are expressed in a variety of tumors and their expression modulates the sensitivity of tumor cells to a wide spectrum of chemotherapeutic agents and γ-irradiation. In the present report, we generated clones of FL5.12 lymphoid cells with similar levels of Bcl-2 and Bcl-XL using the Flag epitope to determine if these survival proteins could provide equivalent protection when challenged with chemotherapy or γ-irradiation. Using four M-phase specific chemotherapeutic agents, Bcl-XL and Bcl-2 provided similar protection against vincristine and vinblastine whereas Bcl-XL afforded as much as 50% greater cell viability than Bcl-2 against etoposide and teniposide-induced cell death. In addition, Bcl-XL provided significantly greater cell viability than Bcl-2 against methotrexate, fluorouracil, and hydroxyurea, three S-phase specific agents. In apoptosis induced by γ-irradiation and cisplatin, two antitumor treatments that are cell-cycle phase-nonspecific agents, both Bcl-XL and Bcl-2 conferred similar protection against γ-irradiation, but Bcl-XL provided better protection than Bcl-2 against cisplatin. These results indicate that Bcl-XL and Bcl-2 confer a differential ability to protect against chemotherapy-induced cell death, which appears to be dependent on the molecular mechanism targeted by the drug rather than its cell-cycle phase specificity.

Acute and long-term lipogenic response to insulin and clenbuterol in bovine intramuscular and subcutaneous adipose tissues.

The present study was conducted to determine whether insulin and clenbuterol affected either short-term (2-h) incubations or long-term (48-h) tissue cultures of i.m. and s.c. adipose tissue explants. Samples were taken from control steers and steers fed 7 mg.head-1.d-1 clenbuterol for 50 d, after which time the drug was withdrawn from the diet for 90 d prior to slaughter. Neither short-term incubations nor long-term explant cultures contained bovine serum albumin (BSA). Insulin (6.67 x 10(-9) M) had no effect (P greater than .05) on lipogenesis in s.c. and i.m. adipose tissue in 2-h tissue incubations of fresh adipose tissue. There was a substantial decrease in activity during the culture period, which was ameliorated somewhat in s.c. adipose tissue by the presence of insulin in the culture media. Clenbuterol exposure for 48 h in vitro decreased the production of lipids from acetate in both adipose tissue depots but had no effect in short-term adipose tissue incubations. Results from the present study confirm that omitting BSA from incubation media does not enhance the responsiveness of bovine s.c. adipose tissue or the less mature i.m. adipose tissue to insulin. Insulin may maintain greater cell viability in 48-h explant cultures.