Claims for Patent: 6,358,737

Summary for Patent: 6,358,737

Title:	Osteocyte cell lines
Abstract:	The present invention relates to a method of producing osteocyte cell line in various stages of differentiation. Such cell line remains stable after more than 20 passages. The osteocyte has a stellate shape with dendritic processes and expresses high level of osteocalcin. More specifically, it provides a method of production for cultured osteocytes of various differentiation stages. Furthermore, it relates to osteocyte cell line, and more specifically cultured osteocyte. The invention also relates to a method for the production of monoclonal antibodies using such cultured osteocytes and further relates to hybridomas and monoclonal antibodies which recognize an osteocyte-specific antigen. Finally, the invention relates to a method of screening for modification factors and binding factors for osteocytes.
Inventor(s):	Bonewald; Lynda F (San Antonio, TX), Kato; Yoichi (Fuji, JP)
Assignee:	Board of Regents, The University of Texas System (TX)
Application Number:	09/152,147
Patent Claims:	1. A method for the production of an osteocyte cell line, said cell line having the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and expresses increased levels of osteocalcin compared to mouse osteoblast or osteoblast-like cell lines, and said method comprising the steps of: (a) isolating bones from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene and preparing bone pieces from the bones; (b) digesting the bone pieces with collagenase solution and harvesting cells into fetal and adult calf serum supplemented medium; and (c) plating the harvested cells and isolating said cell line by selecting a single colony. 2. The method of claim 1, wherein said bone is selected from the group consisting of tibiae, femurs and humeri. 3. The method of claim 1, wherein said medium is alpha-MEM and the supplement is 5% fetal calf serum and 5% adult calf serum. 4. The method of claim 1, wherein said cell line is a mature osteocyte cell line. 5. The method of claim 1, wherein said cell line is a preosteocyte cell line. 6. An immortalized osteocyte cell line which is prepared by the method according to claim 1. 7. A method for the production of an osteocyte cell line, said cell line having the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and expresses increased levels of osteocalcin compared to mouse osteoblast or osteoblast-like cell lines, and said method comprising the steps of: (a) isolating bones from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene and preparing bone pieces from the bones; (b) digesting the bone pieces with collagenase solution and harvesting remaining bone pieces; (c) alternately treating the remaining bone pieces with EDTA and collagenase and harvesting remaining bone pieces; (d) mincing the remaining bone pieces after EDTA and collagenase treatment into smaller chips; (e) culturing the bone chips for a period sufficient to allow migration of cells from the bone chips; (f) harvesting and culturing the migrated cells with fetal and adult calf serum supplemented medium; and (g) isolating said cell line by selecting a single colony. 8. The method of claim 7, wherein said bone is selected from the group consisting of tibiae, femurs and humeri. 9. The method of claim 7, wherein said medium is alpha-MEM and the supplement is 5% fetal calf serum and 5% adult calf serum. 10. The method of claim 7, wherein said cell line is a mature osteocyte cell line. 11. The method of claim 7, wherein said cell line is a preosteocyte cell line. 12. An immortalized osteocyte cell line which is prepared by the method according to claim 7. 13. A method for the production of a cultured osteocyte cell, said cultured osteocyte cell having the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and expresses increased levels of osteocalcin compared to mouse osteoblast or osteoblast-like cell lines, and said method comprising the steps of: (a) isolating bones from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene and preparing bone pieces from the bones; (b) digesting the bone pieces with collagenase solution and harvesting cells; and (c) culturing the harvested cells with fetal and adult calf serum supplemented medium. 14. The method of claim 13, wherein said bone is selected from the group consisting of tibiae, femurs and humeri. 15. The method of claim 13, wherein said medium is alpha-MEM and the supplement is 5% fetal calf serum and 5% adult calf serum. 16. An immortalized osteocyte cell prepared by the method according to claim 13. 17. A method of screening for an osteocyte differentiation modification factor comprising: placing an immortalized osteocyte cell in the presence of a factor to be tested, wherein said osteocyte cell is prepared by the method according to claim 13; measuring a level of a product or activity correlatable with differentiation of the osteocyte expressed by the osteocyte cell in the presence of the factor; and comparing the levels of the expression of the product or activity in the presence and the absence of the factor, wherein a difference in the level of the product or activity in the presence of the factor indicates that the factor is an osteocyte differentiation modification factor. 18. A method of screening for an osteocyte binding factor comprising: placing an immortalized osteocyte cell in the presence of a factor to be tested; and measuring a level of the factor which is bound to the osteocyte cell so as to determine whether the factor is an osteocyte binding factor, wherein said osteocyte cell is prepared by the method according to claim 13. 19. A method for the production of a cultured osteocyte cell, said cultured osteocyte cell having the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and expresses increased levels of osteocalcin compared to mouse osteoblast or osteoblast-like cell lines, and said method comprising the steps of: (a) isolating bones from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene and preparing bone pieces from the bones; (b) digesting the bone pieces with collagenase solution and harvesting remaining bone pieces; (c) alternately treating the remaining bone pieces with EDTA and collagenase and harvesting remaining bone pieces; (d) mincing the remaining bone pieces after EDTA and collagenase treatment into smaller chips; (e) culturing the bone chips for a period sufficient to allow migration of cells from bone chips; and (f) harvesting and culturing the migrated cells with fetal and adult calf serum supplemented medium. 20. The method of claim 19, wherein said bone is selected from the group consisting of tibiae, femurs and humeri. 21. The method of claim 19, wherein said medium is alpha-MEM and the supplement is 5% fetal calf serum and 5% adult calf serum. 22. An immortalized osteocyte cell prepared by the method according to claim 19. 23. A method of screening for an osteocyte differentiation modification factor comprising: placing an immortalized osteocyte cell in the presence of a factor to be tested, wherein said osteocyte cell is prepared by the method according to claim 19; measuring a level of a product or activity correlatable with differentiation of the osteocyte expressed by the osteocyte cell in the presence of the factor; and comparing the levels of the expression of the product or activity in the presence and the absence of the factor, wherein a difference in the level of the product or activity in the presence of the factor indicates that the factor is an osteocyte differentiation modification factor. 24. A method of screening for an osteocyte binding factor comprising: placing an immortalized osteocyte cell in the presence of a factor to be tested; and measuring a level of the factor which is bound to the osteocyte cell so as to determine whether the factor is an osteocyte binding factor, wherein said osteocyte cell is prepared by the method according to claim 19. 25. An immortalized mouse osteocyte cell line, said cell line being obtained from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene, wherein said cell line has the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and increased levels of osteocalcin expression compared to mouse osteoblast or osteoblast-like cell lines. 26. The immortalized cell line of claim 25, wherein said cell line overexpresses T-antigen protein. 27. An immortalized mouse osteocyte cell or a cell obtained by passaging said mouse osteocyte cell having the following properties: available to passage more than 20 times, a stellate shape with dendritic processes and increased levels of osteocalcin expression compared to mouse osteoblast or osteoblast-like cell lines, wherein said osteocyte cell is obtained from a transgenic mouse whose genome contains an osteocalcin promoter driven T-antigen transgene. 28. The immortalized osteocyte cell of claim 27, wherein said osteocyte cell overexpresses T-antigen protein. 29. A method of screening for an osteocyte differentiation modification factor comprising: placing an osteocyte cell of claim 27 in the presence of a factor to be tested; measuring a level of a product or activity correlatable with differentiation of the osteocyte expressed by the osteocyte cell in the presence of the factor; and comparing the levels of the expression of the product or activity in the presence and the absence of the factor, wherein a difference in the level of the product or activity in the presence of the factor indicates that the factor is an osteocyte differentiation modification factor. 30. The method of screening for an osteocyte modification factor according to claim 29, wherein said osteocyte cell overexpresses T-antigen protein. 31. A method of screening for an osteocyte binding factor comprising: placing an osteocyte cell of claim 27 in the presence of a factor to be tested; and measuring a level of the factor which is bound to the osteocyte cell so as to determine whether the factor is an osteocyte binding factor. 32. The method of screening for an osteocyte binding factor according to claim 31, wherein said osteocyte cell overexpresses T-antigen protein. 33. An osteocyte cell line, MLO-Y4 (ATCC accession No. CRL-12161) or a passaged cell line thereof.

Details for Patent 6,358,737

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2016-07-31