OBJECTIVETo fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions.

METHODSWe chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis.

RESULTSCells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05).

CONCLUSIONSThe cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.

Alginates ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; physiopathology ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Glucuronic Acid ; Hexuronic Acids ; Humans ; Lung Neoplasms ; metabolism ; pathology ; physiopathology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Printing, Three-Dimensional ; Sepharose ; Spheroids, Cellular ; pathology ; Time Factors ; Tissue Scaffolds ; Tumor Microenvironment

BACKGROUND: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA). METHODS: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining. RESULTS: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases. CONCLUSIONS: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.
Biopsy, Fine-Needle ; Diagnosis ; Immunohistochemistry ; Paraffin ; Paraffin Embedding ; Sepharose*

BACKGROUND: Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. METHODS: Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. RESULTS: The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. CONCLUSIONS: Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks.
Gels ; Humans ; Paraffin ; Sepharose ; Tissue Array Analysis ; Tissue Donors

PURPOSE: Although the etiology of breast cancer is multifactorial, oxidative stress plays an important role in carcinogenesis. In this study, manganese superoxide dismutase (MnSOD) gene polymorphism and activity were evaluated in benign and breast cancer tissue. METHODS: One hundred and one females were enrolled in this study, 65 who were histopathologically diagnosed with breast cancer and 46 who were benign patients. MnSOD enzyme activity was determined using an indirect competitive inhibition assay and MnSOD gene polymorphism using poly merase chain reaction and agarose gel electrophoresis. RESULTS: MnSOD enzymatic activity (79.83+/-42.14) was lower in breast cancer tissue compared to benign tumors (236.18+/-46.37). At the same time, MnSOD enzymatic activity among Ala/Val patients was significantly lower in breast cancer tissue (39.19+/-7.33) than in Val/Val malignant breast tumors tissue (96.9+/-22.9). MnSOD enzymatic activity was significantly lower in Val/Val cancer tissue (96.9+/-22.9) than in benign tissue (255.44+/-42.7). CONCLUSION: Breast cancer tumors contain less MnSOD than benign breast samples. Patients with Ala/Val polymorphism had reduced MnSOD activity compared to patients with Val/Val breast cancer. Ala/Val gene polymorphism may be a risk factor associated with more advanced breast cancer stage. MnSOD gene polymorphism Ala/Val may be a risk factor associated with more advanced breast cancer stage, and reduction of MnSOD activity may be a mechanism of the progression of benign to malignant tumors. Further investigations are needed to evaluate the role of MnSOD in breast cancer progression.
Breast ; Breast Neoplasms ; Female ; Humans ; Manganese ; Oxidative Stress ; Risk Factors ; Sepharose ; Superoxide Dismutase

OBJECTIVES. The goal of the study is to find a reasonable aIternative test that can be utilized in the Philippine setting to operationalize the Universal Newborn Hearing Screening Act. Thus the components of the Voice Test were studied. The objectives of the study are to determine: (1) which of the two words "Baah" and "Psst" is better for newborn hearing screening rocedure as far as their physical characteristics are concerned, ~) how do the two words "Baah" and Psst" differ between genders and distance from sound source, (3) to determine the proportion of the participants who could recite the words at intensity of 80db or louder.

METHODS. Frequency characteristics and sound intensity differences of two words "Baah" and "Psst" were determined and ompared.

RESULTS. The word "Baah" exhibited more favorable physical attributes over the word 'Psst" for purposes of being a screening tool for newborn hearing assessment.

CONCLUSION. This study reports the results of an initial step in the search for an inexpensive, feasible, and valid tool for neonatal earing screening. Correlation studies with speech developmental milestones may eventually enhance the usefulness of the voice test.

Human ; Male ; Female ; Aged ; Middle Aged ; Adult ; Young Adult ; Adolescent ; Neonatal Screening ; 1012-s-acetamide Adipic Hydrazide Sepharose 4b ; Benzodiazepines ; Sepharose

Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications.
Adhesives ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Cues ; Hydrogel ; Neurites ; Neurons ; Nylons ; Rats ; Sepharose ; Tissue Engineering ; Weights and Measures

In this paper, a kind of skin dressing, agarose- grafting- hyaluronic acid (Ag-g-HA) sponge was applied to test the modified agarose based scaffold for skin regeneration. The bFGF loading agarose-grafting hyaluronan scaffold had homogenous porosities, and the loaded bFGF was bioactive in 2 weeks. The Ag-g-HA sponge was applied into skin of mice, and it was found that the dressing promoted skin regeneration and no infection and leakage in lesion site took place. H&E staining results showed that the repaired skin was similar to autologous skin. These demonstrate that Ag-g-HA sponge has a promise in skin regeneration.
Animals ; Bandages ; Female ; Fibroblast Growth Factor 2 ; physiology ; Hyaluronic Acid ; therapeutic use ; Mice ; Mice, Inbred C57BL ; Polysaccharides ; isolation & purification ; therapeutic use ; Random Allocation ; Seaweed ; chemistry ; Sepharose ; isolation & purification ; therapeutic use ; Surgical Sponges ; Wound Healing ; drug effects ; Wounds and Injuries ; therapy

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.
Chromatography ; Deoxyribonucleases ; Haemonchus ; Intestines ; Proteins ; Sepharose

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.
Chromatography ; Deoxyribonucleases ; Haemonchus ; Intestines ; Proteins ; Sepharose

OBJECTIVETo assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound.

METHODSTargeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images.

RESULTSThe VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05).

CONCLUSIONMBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.

Antibodies, Monoclonal ; chemistry ; immunology ; Contrast Media ; chemistry ; Humans ; Integrin alphaVbeta3 ; immunology ; metabolism ; Microbubbles ; Sepharose ; Thrombosis ; diagnostic imaging ; Ultrasonography