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Last Updated: April 25, 2024

Claims for Patent: 9,856,454

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Summary for Patent: 9,856,454

Title:	Rapid mincing of adipose tissues to isolate live cells in vitro
Abstract:	A method for rapid mincing of adipose tissues to isolate live cells in vitro is disclosed to overcome the drawbacks of a lead procedure of isolating live cells by holding a knife to mince adipose tissues, which can only isolate a small number of live cells and obtain low cell viability. The method includes: providing an adipose tissue; mincing the adipose tissue homogeneously by a mincing device; adding a reagent into the minced adipose tissue to perform hydrolysis; performing centrifuge and isolation; and removing a supernatant to obtain a cell pellet. Therefore, the time of mincing adipose tissues can be shortened, and contaminations caused by repeated use of the knife can be avoided. The method can be used for isolating live cells of adipose tissues to improve the number of live cells per unit weight of adipose tissues without reducing the cell viability.
Inventor(s):	Sun; Li-Yi (Hualien County, TW), Li; Dian-Kun (Hualien County, TW), Pang; Cheng-Yoong (Hualien County, TW), Chang; Yao-Jen (Hualien County, TW)
Assignee:	Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (Hualien, TW)
Application Number:	14/191,675
Patent Claims:	1. A method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues, comprising the steps as follows: step (a) providing an adipose tissue; step (b) arranging the adipose tissue into an enclosed vessel of a cutting device, wherein the cutting device is a disposable homogenizer comprising a power unit; step (c) applying an acting force on the enclosed vessel by the cutting device to enable a preset cutting tool in the enclosed vessel to implement rapid and homogeneous mincing on the adipose tissue in a closed environment, thereby obtaining a homogenized adipose tissue, wherein a relative centrifugal force of the cutting tool is 16-25.times.g; step (d) adding a reagent into the homogenized adipose tissue to implement a hydrolysis reaction; step (e) filtering and centrifugally separating the homogenized adipose tissue treated via the hydrolysis reaction; and step (f) removing a supernate after the centrifugal separation so as to obtain a cell pellet, which contains isolated stromal vascular fraction (SVF) cells. 2. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein a step of cleaning the adipose tissue via a phosphate buffer solution is included before step (a). 3. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein the adipose tissue in step (a) is a lump adipose tissue obtained from surgery excision of a mammal. 4. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 3, wherein the mammal is a human being. 5. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein the disposable homogenizer comprises a disposable enclosed vessel, and the inside of the disposable enclosed vessel is provided with a cutting tool. 6. The method for isolating live, stromal vascular fraction (SVF) adipose tissues according to claim 5, wherein the homogeneous mincing in step (c) is performed by arranging 1 g to 6 g of adipose tissues into the disposable enclosed vessel with a volume of 15 ml to implement rapid and homogeneous mincing. 7. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 6, wherein the homogeneous mincing in step (c) is performed by arranging 3 g of adipose tissues into the disposable enclosed vessel with a volume of 15 ml to implement rapid and homogeneous mincing. 8. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 7, wherein an acting time of the power unit is 3-10 minutes. 9. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein the reagent in step (d) is selected from the group consisting of a trypsin, a dispase, a gelatase, a hyaluronidase, a collagenase type I, a collagenase type IV and combinations thereof. 10. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein the hydrolysis reaction in step (d) is implemented in a constant temperature hybridization reaction oven for the whole process under conditions of 4-45 DEG C., 5 rpm-50 rpm and 0.5 h-24 h. 11. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 10, wherein the preferred conditions are 37 DEG C., 15 rpm and 8 h. 12. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein the conditions for centrifugation in step (e) are 400.times.g and 10 minutes. 13. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein step (e) comprises a step of moving a filtered fluid obtained after filtering the homogenized adipose tissue treated via the hydrolysis reaction into a centrifugal tube, so as to implement centrifugal separation. 14. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 1, wherein after step (f), performing a step (g) of adding the phosphate buffer solution into the cell pellet, cleaning the cell pellet, and then centrifuging again so as to remove a supernate comprising blood and the reagent to obtain a cleaned cell pellet. 15. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 14, wherein after being cleaned in step (g), a step (h) of arranging the cell pellet into a culture medium for culturing so as to obtain amplified cells. 16. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 15, wherein the culture medium comprises a basal culture medium (IMDM), a serum additive and a fibroblast growth factor-2 (FGF-2). 17. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 16, wherein the serum additive is a fetal bovine serum with a volume percentage concentration of 2% to 10%, and a concentration of the FGF-2 is 1 ng/ml to 20 ng/ml. 18. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 15, wherein the amplified cell is an adipose tissue-derived stem cell which is substantially undifferentiated. 19. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 18, wherein the adipose tissue-derived stem cell at least shows a cell surface antigen CD90, or CD 105 or a combination thereof, and does not show CD45. 20. A method for cell therapy, regenerative medicine or cell and tissue engineering, comprising; making an adipose tissue-derived stem cell according to the method of claim 19; and applying the adipose tissue-derived stem cells singly or in combination as a material for cell therapy, regenerative medicine or cell and tissue engineering. 21. A method for treating one or a combination of degenerated diseases, tissue damage repair, organ regeneration or tissue regeneration, comprising; making an adipose tissue-derived stem cell according to the method of claim 18; and preparing medicinal compositions for cell therapy from the adipose tissue-derived stem cell. 22. A method for establishing a cell bank, comprising; making an amplified adipose tissue-derived stem cells by the method according to claim 18; and implementing cryopreservation on the amplified adipose tissue-derived stem cell. 23. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 18, wherein a step (i') of implementing induced differentiation on the amplified adipose tissue-derived stem cell so as to obtain a cell differentiated from the amplified adipose tissue-derived stem cell is included after step (h). 24. The method for isolating live, stromal vascular fraction (SVF) cells in vitro from adipose tissues according to claim 23, wherein the cells differentiated from the amplified adipose tissue-derived stem cells include one of an osteogenic cell, an adipose cell or a cartilage cell.

Details for Patent 9,856,454

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Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	05/05/2004	⤷ Try a Trial	2039-02-26
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	12/02/2004	⤷ Try a Trial	2039-02-26
Amphastar Pharmaceuticals, Inc.	AMPHADASE	hyaluronidase	Injection	021665	10/26/2004	⤷ Try a Trial	2039-02-26
Akorn, Inc.	HYDASE	hyaluronidase	Injection	021716	10/25/2005	⤷ Try a Trial	2039-02-26
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965	⤷ Try a Trial	2039-02-26
>Applicant	>Tradename	>Biologic Ingredient	>Dosage Form	>BLA	>Approval Date	>Patent No.	>Expiredate

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