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1.
Biomater Sci ; 9(19): 6574-6583, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34582534

RESUMO

Porphyromonas gingivalis, the pathogen of periodontal disease, is thought to be involved in various diseases throughout the body via gingival tissue blood capillaries. However, the dynamic analysis of the infection mechanism, particularly the deep invasion process of the gingival tissue, has not yet been elucidated because of the lack of both in vivo and in vitro models. In this study, we developed a vascularized three-dimensional (3D) gingival model with an epithelial barrier expressing cell-cell junctions using collagen microfibers (CMFs) to enable the dynamic analysis of the P. gingivalis invasion process. Lipid raft disruption experiments in the gingival epithelial cell layer demonstrated that P. gingivalis migrates into the deeper epithelium via the intercellular pathway rather than intracellular routes. P. gingivalis was shown to invade the 3D gingival model, being found inside blood capillaries during two days of culture. Notably, the number of bacteria had increased greatly at least two days later, whereas the mutant P. gingivalis lacking the cysteine proteases, gingipains, showed a significantly lower number of survivors. The secretion of interleukin-6 (IL-6) from the gingival tissue decreased during the two days of infection with the wild type P. gingivalis, but the opposite was found for the mutant suggesting that P. gingivalis infection disturbs IL-6 secretion at an early stage. By allowing the dynamic observation of the P. gingivalis invasion from the epithelial cell layer into the blood capillaries for the first time, this model will be a powerful tool for the development of novel therapeutics against periodontal infection related diseases.


Assuntos
Capilares , Porphyromonas gingivalis , Células Cultivadas , Células Epiteliais , Gengiva , Humanos
2.
Nat Commun ; 12(1): 5059, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429413

RESUMO

With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.


Assuntos
Bioimpressão/métodos , Géis , Carne , Tendões , Animais , Bovinos , Técnicas de Cultura de Células , Colágeno , Células Endoteliais , Músculos/citologia , Músculos/fisiologia , Impressão Tridimensional , Células-Tronco , Tendões/citologia , Engenharia Tecidual
3.
Curr Protoc Cell Biol ; 88(1): e112, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32776707

RESUMO

Construction of organized three-dimensional (3D) tissue with extracellular matrix (ECM) and multiple types of cells is important for tissue engineering to enable tissue function and enhance cellular function. However, the concentration of ECM and the thickness of the 3D tissue have been limited in previous methods due to a lack of permeability to nutrients and oxygen. Besides, it is difficult to use matured natural ECM as a cell scaffold without chemical modification due to its insolubility. In this article, we focus on multi-layered structure, which is commonly found in living tissue such as skin, blood vessels, and other organs. Here, we describe the preparation of a paper-like scaffold (ECM paper) from micro-fibered natural ECM and the construction of 3D multi-layered tissue composed of cell layers and ECM layers by stacking cell-seeded ECM papers. The thickness and components of the ECM layers are easily controllable by changing the composition of the ECM papers, and the fibrous structure of ECM paper shows high permeability and permits cell migration. Additionally, the ECM microfiber, which is physically defiberized from natural ECM, has a high ECM concentration equal to that of living tissue and high stability under physiological conditions. Therefore, this set of protocols enables construction of multi-layered 3D tissue composed of precisely controlled ECM layers and cell layers that may contribute to the assembly of tissue models. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of extracellular matrix paper Basic Protocol 2: Evaluation of cellular function of cells on extracellular matrix paper Basic Protocol 3: Construction of multi-layered 3D tissue.


Assuntos
Movimento Celular , Matriz Extracelular , Engenharia Tecidual , Tecidos Suporte , Animais , Células Cultivadas , Matriz Extracelular/química , Humanos , Modelos Biológicos , Engenharia Tecidual/métodos , Tecidos Suporte/química
4.
Adv Biosyst ; 4(5): e2000038, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32402125

RESUMO

Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.

5.
Sci Rep ; 9(1): 11346, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383871

RESUMO

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS. After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.


Assuntos
Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Proteínas Proto-Oncogênicas p21(ras)/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Linhagem Celular Tumoral , Códon/efeitos dos fármacos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , DNA de Forma B/efeitos dos fármacos , DNA de Forma B/genética , Detecção Precoce de Câncer , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Nylons/química , Nylons/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirróis/química , Pirróis/farmacologia
6.
Acta Biomater ; 84: 194-207, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30502481

RESUMO

Although adipose tissue is one of the most abundant tissues of the human body, its reconstruction remains a competitive challenge. The conventional in vitro two or three-dimensional (2D or 3D) models of mature adipocytes unfortunately lead to their quick dedifferentiation after one week, and complete differentiation of adipose derived stem cells (ADSC) usually requires more than one month. In this context, we developed biomimetic 3D adipose tissues with high density collagen by mixing type I collagen microfibers with primary mouse mature adipocytes or human ADSC in transwells. These 3D-tissues ensured a better long-term maintained phenotype of unilocular mature adipocytes, compared to 2D, with a viability of 96 ±â€¯2% at day 14 and a good perilipin immunostaining, - the protein necessary for stabilizing the fat vesicles. For comparison, in 2D culture, mature adipocytes released their fat until splitting their single adipose vesicle into several ones with significantly 4 times smaller size. Concerning ADSC, the adipogenic genes expression in 3D-tissues was found at least doubled throughout the differentiation (over 8 times higher for GLUT4 at day 21), along with it, almost 4 times larger fat vesicles were observed (10 ±â€¯4 µm at day 14). Perilipin immunostaining and leptin secretion, the satiety protein, attested the significantly doubled better functionality of ADSC in 3D adipose tissues. These obtained long-term maintained phenotype and fast adipogenesis make this model relevant for either cosmetic/pharmaceutical assays or plastic surgery purposes. STATEMENT OF SIGNIFICANCE: Adipose tissue has important roles in our organism, providing energy from its lipids storage and secreting many vital proteins. However, its reconstruction in a functional in vitro adipose tissue is still a challenge. Mature adipocytes directly extracted from surgery liposuctions quickly lose their lipids after a week in vitro and the use of differentiated adipose stem cells is too time-consuming. We developed a new artificial fat tissue using collagen microfibers. These tissues allowed the maintenance of viable big unilocular mature adipocytes up to two weeks and the faster adipogenic differentiation of adipose stem cells. Moreover, the adipose functionality confirmed by perilipin and leptin assessments makes this model suitable for further applications in cosmetic/pharmaceutical drug assays or for tissue reconstruction.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Colágeno/química , Tecidos Suporte/química , Adipócitos/citologia , Tecido Adiposo/citologia , Animais , Regulação da Expressão Gênica , Humanos , Leptina/biossíntese , Camundongos
7.
Cancer Sci ; 107(7): 936-43, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27116474

RESUMO

In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence.


Assuntos
Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reprodutibilidade dos Testes
8.
PLoS One ; 8(5): e62989, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23671647

RESUMO

BACKGROUND: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC). In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC). For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS) for point of care testing (POCT). Here we report the results of a comparison study between AMDS and direct sequencing (DS) in the detection of KRAS, BRAF and PI3KCA somatic mutations. METHODOLOGY/PRINCIPAL FINDING: DNA was extracted from a slice of either frozen (n = 89) or formalin-fixed and paraffin-embedded (FFPE) CRC tissue (n = 70), and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples) detected by DS were also successfully (100%) detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous "hot spot" mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89) and 74.3% (52/70) in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41) were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Mutação , Patologia Molecular/métodos , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA/métodos , Formaldeído/química , Secções Congeladas , Humanos , Inclusão em Parafina/métodos , Patologia Molecular/instrumentação , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fixação de Tecidos/métodos , Proteínas ras/genética
9.
Anal Biochem ; 408(2): 197-205, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20850410

RESUMO

Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET-PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET-PHFA detected KRAS mutations (1.25-50%) from all cell line DNA titration samples.


Assuntos
Análise Mutacional de DNA/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Reação em Cadeia da Polimerase , Proteínas ras/genética
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