Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
  • G01N2333/43595—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from coelenteratae, e.g. medusae

Definitions

• the present invention relates to the field of engineered cell lines.
• tissue engineering This discipline is the sum of knowledge in the fields of cell biology, molecular biology, histology and biomedical and biomaterial technologies.
• the enormous progress achieved in biology and biomaterial technology is probably one of the main factors responsible for development of tissue engineering.
• the time of patient inactivity may be extremely long, with unavoidable negative effects on both patient life quality and in terms of social costs.
• bone tissue engineering has special importance from both the biological and clinical point of view.
• Tissue engineering was initially directed at the study of biocompatible implantable materials, which did not cause "noticeable adverse reactions" at the implant site in animal models or volunteer patients. More recently, this discipline has been directed at the study of materials capable of stimulating cell proliferation and thus regeneration of the tissue temporarily replaced by such material. In this context, it is evident that said materials must allow cell proliferation and implant, but at the same time must not affect the histological characteristics of adherent cells, thus ensuring restoration of a tissue that is functionally and structurally identical to the physiological tissue. The possibility to evaluate such properties, from both a quantitative and qualitative point of view, represents a crucial step to characterize and select the best suited biomaterials for clinical applications. Until now, these evaluations involved an initial use of animal models and ultimately human volunteers, hence a very lengthy data collection period.
• the graph in Figure 1 shows the linear relationship existing between fluorescence emission and number of Saos-EGFP cells in each sample.
• the present invention offers a solution to the lack of experimental cellular models, with histological characteristics of bone tissue, that can be easily identified and quantified following adhesion to biocompatible materials.
• This is realized by the use of cells, hereinafter termed Saos-EGFP, derived from the Saos-2 human osteosarcoma cell line, engineered with the coding portion of the enhanced green fluorescent protein (EGFP) gene.
• the Saos-EGFP cell line according to the invention is a hybrid cell line derived by integration of genetic information from organisms belonging to different taxonomic phyla, wherein a jellyfish protein is expressed in a human substrate.
• Saos-EGFP cells according to the present invention have histological properties typical of human osteoblasts and constitutively express EGFP.
• the enhanced EGFP form was obtained by mutagenesis (Cormack et al., 1996) of parental
• GFP isolated from Aequorea victoria jellyfish (Prasher et al., 1992).
• the GFP gene drives expression of the fluorescent protein also in heterologous cellular systems, without inducing any toxicity.
• the fluorescence emitted by Saos-EGFP cells enables visualization of the cellular behaviour on the studied materials by fluorescence microscopy .
• the fluorescence intensity of a sample provides an indirect indication of the number of cells present in the sample.
• the cells herein employed are derived from the Saos-2 cell line that was originated from a human osteosarcoma and however maintains the osteoblastic properties of the tissue of origin (Rodan et al., 1987). Cells were cultured in monolayer at 37 0 C in DMEM medium supplemented with 10% Fetal Bovine Serum. Saos-2 cells were transfected with 5 ug of pCMV-EGFP construct that was made in the laboratory of the applicants by use of a known technique (Morelli et al., 2004). Said construct contains the gene for enhanced green fluorescent protein (EGFP) under control of the herpetic Human Cytomegalovirus (HCMV) early promoter and the neo gene encoding the resistance to Geneticin or G418.
• EGFP enhanced green fluorescent protein
• HCMV herpetic Human Cytomegalovirus
• Resistant Saos-EGFP clones were selected by growth in medium containing an appropriate amount of Geneticin. Said clones have been isolated and expanded as independent lines. Said lines constitutively express the fluorescent EGFP protein.
• Immunofluorescence The histological characteristics of Saos-EGFP cells have been tested by immunofluorescence assays for osteopontin and osteonectin, that are specific protein markers of bone tissue. The cytoskeletal organization of the cells has been revealed by visualization of actin fibres upon staining with fluorochrome-conjugated phalloidin . For immunofluorescence experiments, parental Saos-2 and engineered Saos-EGFP cells have been grown for 18 hours on 12 mm diameter glass slides.
• the invention allows direct visualization of cells adhering to biocompatible materials by observation under a fluorescence microscope. This allows evaluation of the ability of cells to colonize the support, providing also important indications about their mode of distribution. This information is particularly required in order to determine the potential for restoration of a highly organized tissue such as bone. Table 1. Determination of cell number

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• Proteomics, Peptides & Aminoacids (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Cited By (1)

• 2005
  • 2005-03-11 IT ITFI20050038 patent/ITFI20050038A1/it unknown
• 2006
  • 2006-03-09 WO PCT/EP2006/060603 patent/WO2006095011A1/en not_active Application Discontinuation

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