C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 5/10 (2006.01) C12N 5/08 (2006.01)
Patent
CA 2324479
An human keratinocyte cell line immortalized by means of at least one functional tumor gene of retroviral origin characterised in that it is (1) non-tumorigenic, (2) conserves the capacity for differentiation and for the expression of proteins and of enzymes expressed by normal differentiated keratinocytes even after an elevated number of passages in culture, and (3) forms a stratified and polarised epithelium having a stratum corneum ortho- keratinocyte, if cultivated in an organo-typical culture in a medium without serum and without a layer of nourishing cells. An improved process to immortalize human skin cells to obtain immortalized keratinocytes is characterised in that it comprises the following steps: (i) isolating a sample of human skin; (ii) preparing the sample of skin for in vitro culture; (iii) obtaining keratinocytes from this prepared sample of human skin and seeding in a growth medium without serum with said keratinocytes, on culture plates provided with a coating comprising fibronectin, collagen type I and SAB, which facilitate the fixation and the growth of cells; (iv) replacing the medium in a way sufficient to obtain an optimal confluent growth of cells in culture, continuously maintaining the coating of the plate; (v) transferring keratinocytes into a selection medium without serum on culture plates coated previously in a similar manner; (vi) infecting keratinocytes using functional tumor genes of at least two different retrovirus; (vii) transferring the resulting immortalized keratinocytes to a proliferation medium suitable for the proliferation of immortalised keratinocytes on culture plates coated in a similar way; (viii) transferring the resulting proliferated keratinocytes into a differentiation medium having a high content of calcium on culture boxes coated in a similar way. Use of keratinocytes for immunological, pharmacological, photo- and chemical- toxigological analyses of skin reaction and for expression of heterologous genes and for the construction of artificial skin.
Lignée cellulaire de kératinocytes humains immortalisés au moyen d'au moins un gène tumorigène fonctionnel provenant d'un rétrovirus caractérisée en ce qu'elle est (1): non-tumorigénique; (2) conserve l'aptitude à la différenciation et à l'expression de protéines et d'enzymes qui sont exprimées par les kératinocytes différenciés normaux même après un taux élevé de passages en culture de tissus; et (3) forme un épithélium stratifié et polarisé comportant un stratum corneum ortho-kératosique, lorsqu'elle est cultivée en culture organotypique dans un milieu sans sérum et sans couche de cellules nourricières. Procédé perfectionné pour immortaliser des cellules cutanées humaines afin d'obtenir des kératinocytes immortalisés, caractérisé en ce qu'il comprend l'étape d'infection des kératinocytes en utilisant des gènes tumorigènes fonctionnels d'au moins deux rétrovirus différents. Utilisation des kératinocytes selon l'invention pour des analyses immunologiques, pharmacologiques, photo- et chimiotoxicologiques de réaction cutanée, pour l'expression de gènes hétérologues, et pour la construction d'une peau artificielle.
Borden Ladner Gervais Llp
Societe Des Produits Nestle S.a.
LandOfFree
Immortalised cell line derived from normal human skin tissues does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Immortalised cell line derived from normal human skin tissues, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Immortalised cell line derived from normal human skin tissues will most certainly appreciate the feedback.
Profile ID: LFCA-PAI-O-1357578