Claims for Patent: 9,807,986

Summary for Patent: 9,807,986

Title:	Methods for establishing animal model of hepatocellular carcinoma bone metastasis
Abstract:	A method for establishing an animal model of hepatocellular carcinoma (HCC) bone metastasis, the method including: 1) establishing 97H and LM3 cell clones with stable expression of firefly luciferase (LUC); 2) allowing HCC cells to form bone metastasis in nude mice via intratibial injection; 3) reproducing HCC bone metastasis in nude mice via intracardiac injection of tumor cells; and 4) isolating a sub-population of tumor cells that targets metastasis to bone. The 97H and the LM3 are highly metastatic HCC cell lines transfected with luciferase gene. BALB/cA-nu mice are 4-5 weeks old and maintained in laminar flow cabinets under SPF conditions and received human care throughout an entire study. A cell number for intratibial injection is 0.5.times.10.sup.6, and a cell number for intracardiac injection is 1.times.10.sup.6.
Inventor(s):	Hou; Rui (Wuhan, CN), Wang; Yuwei (Wuhan, CN), Liang; Huifang (Wuhan, CN), Zhang; Zhanguo (Wuhan, CN), Liu; Zhimin (Wuhan, CN), Zhang; Binhao (Wuhan, CN), Zhang; Bixiang (Wuhan, CN), Chen; Xiaoping (Wuhan, CN)
Assignee:
Application Number:	15/083,269
Patent Claims:	1. A method for establishing an animal model of hepatocellular carcinoma (HCC) bone metastasis, the method comprising: 1) transfecting 97H and LM3 cell clones with luciferase genes to yield 97H/Luc+ and LM3/Luc+ cells expressing firefly luciferase (LUC); 2) intracardially injecting the 97H/Luc+ and LM3/Luc+ cells into each nude mouse of a set of nude mice to induce the formation of metastatic 97H/Luc+ and LM3/Luc+ cells in bones of the set of nude mice, wherein the nude mice are 4-5-week-old BALB/cA-nu mice; and 3) isolating the metastatic 97H/Luc+ and LM3/Luc+ cells obtained in 2) from the bones of the set of nude mice. 2. The method of claim 1, wherein 2) comprises: a) pelleting the 97H/Luc+ and LM3/Luc+ cells to yield a cell pellet comprising the 97H/Luc+ and LM3/Luc+ cells; and washing the cell pellet several times in sterile PBS; b) suspending the cell pellet obtained in a) in an ice-cold FBS-free DMEM medium by pipetting the cell pellet up and down to yield a cell suspension having a concentration of 0.5.times.10.sup.7 cells/ml; and filtering the cell suspension through a 200-mesh screen to yield a single cell suspension of the 97H/Luc+ and LM3/Luc+ cells, wherein the single cell suspension is a cell suspension comprising single cells of the 97H/Luc+ and LM3/Luc+ cells; c) anaesthetizing each nude mouse of the set of nude mice using isoflurane gas anesthesia to obtain a set of anaesthetized nude mice; d) fixing each nude mouse of the set of anaesthetized nude mice in a supine position on a sterile surface with the head of each nude mouse of the set of anaesthetized nude mice in a nozzle which supplies isoflurane at a maintenance dose of 2%; e) aspirating the single cell suspension obtained in b) into a 29-G needle syringe for each nude mouse of the set of anaesthetized nude mice, and avoiding producing air bubbles in the 29-G needle syringe; f) inserting the needle of the 29-G needle syringe through the diaphragm of each nude mouse of the set of anaesthetized nude mice approximately 3 mm to the left of the sternum of each nude mouse of the set of anaesthetized nude mice and positioning the needle of the 29-G needle syringe toward the heart of each nude mouse of the set of anaesthetized nude mice; g) advancing the needle of the 29-G needle syringe into the left ventricle of each nude mouse of the set of anaesthetized nude mice; h) injecting 100 .mu.l of the single cell suspension into the left ventricle of each nude mouse of the set of anaesthetized nude mice over a period of 30 seconds by the 29-G needle syringe; and i) monitoring development and progression of the metastatic 97H/Luc+ and LM3/Luc+ cells weekly by monitoring LUC expression of the 97H/Luc+ and LM3/Luc+ cells in the bones of the set of nude mice using bioluminescence imaging (BLI). 3. The method of claim 1, wherein 3) comprises: i) identifying bones comprising the metastatic 97H/Luc+ and LM3/Luc+ cells by identifying LUC expression of the 97H/Luc+ and LM3/Luc+ cells in bones of the set of nude mice using bioluminescence imaging (BLI); ii) euthanizing the set of nude mice obtained in 2) to obtain euthanized nude mice; iii) resecting the bones comprising the metastatic 97H/Luc+ and LM3/Luc+ cells from bodies of the euthanized nude mice and removing surrounding soft tissues of the bones to yield resected bones; iv) mincing the resected bones to yield minced bones; v) adding RBS lysis buffer and collagenase/hyaluronidase digesting solution to the minced bones to yield a mixture comprising the minced bones, the RBS lysis buffer, and the collagenase/hyaluronidase digesting solution; vi) incubating the mixture obtained in v) on a rocking plate and then centrifuging the mixture to yield a metastatic cell pellet comprising the metastatic 97H/Luc+ and LM3/Luc+ cells; vii) re-suspending the metastatic cell pellet obtained in vi) in 10% FBS DEME with 200 .mu.g/ml G418 to yield a metastatic cell suspension; viii) seeding the metastatic cell suspension onto a 12-well plate; and ix) renewing the 10% FBS DEME with 200 .mu.g/ml G418 after 24 hours.

Details for Patent 9,807,986

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	05/05/2004		2039-02-26
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	12/02/2004		2039-02-26
Amphastar Pharmaceuticals, Inc.	AMPHADASE	hyaluronidase	Injection	021665	10/26/2004		2039-02-26
Akorn, Inc.	HYDASE	hyaluronidase	Injection	021716	10/25/2005		2039-02-26