Claims for Patent: 9,839,654

Summary for Patent: 9,839,654

Title:	Isolation, expansion and characterization of precursor/stem cells from dental tissues
Abstract:	A method for isolating and proliferating at least one type of precursor cell from dental origin from a single donor includes the following isolating the precursor cells from dental origin of a single donor in a sample collection media and preparing a primary stock culture. The primary stock culture is proliferated sequentially to obtain first, second and third sub-cultured stocks with cell counts ranging between 5.times.10.sup.6 cells and 10.times.10.sup.6 cells, 20.times.10.sup.6 cells and 400.times.10.sup.6 cells, 150.times.10.sup.6 and 300.times.10.sup.6 cells respectively. The precursor cells from the third subculture are harvested and cryo-preserved to obtain a precursor cell population for cell transplantation. The dental origin of the precursor cells is from pulp, apical papilla or periodontal ligament. The precursor cell originates from mesenchymal stem cells, ecto-mesenchymal cells, neural stem cells, dental progenitor cells or CD117.sup.+ cells.
Inventor(s):	Govindasamy; Vijayendran (Putrajaya, MY), Kathivaloo; Premasangery (Putrajaya, MY)
Assignee:	HYGIEIA INNOVATION SDN BHD (Putrajaya, MY)
Application Number:	14/831,794
Patent Claims:	1. A method for isolating and proliferating at least one type of precursor cell from dental origin from a single donor, said method comprising the following steps: a) isolating the at least one type of precursor cell from dental origin of a single donor in a sample collection media and preparing a primary stock culture, wherein the sample collection media comprises DMEM media in an amount ranging between 66.5% and 90.5%, Human Albumin Phosphate in an amount ranging between 5% and 20%, Penicillin-Streptomycin in an amount ranging between 1% and 3%, fungizone in an amount ranging between 1% and 3%, and insulin-transferrin-selenium in an amount ranging between 2.5% and 7.5%; b) proliferating the primary stock culture sequentially to obtain a first, second and third sub-cultured stocks with cell counts ranging between 5.times.10.sup.6 cells and 10.times.10.sup.6 cells, 20.times.10.sup.6 cells and 400.times.10.sup.6 cells, 150.times.10.sup.6 and 300.times.10.sup.6 cells respectively; and c) harvesting and cryo-preserving the at least one type of precursor cell from the third subculture to obtain a precursor cell population capable of being used for cell transplantation, wherein the dental origin of the precursor cell is at least one selected from the group consisting of pulp, apical papilla, and periodontal ligament, and wherein the at least one type of precursor cell is at least one selected from the group consisting of mesenchymal stem cells, ecto-mesenchymal cells, neural stem cells, dental progenitor cells, and CD117+ cells. 2. The method as claimed in claim 1, wherein the method step of preparing the primary stock culture from the at least one type of precursor cell comprises steps of: a. mincing a dental tissue to obtain a desired size ranging between 0.5 mm.sup.3 and 2.5 mm.sup.3; b. digesting the minced dental tissue by incubating it in a mixture of Collagenase Type-IV Stock Solution and Knockout-Dulbecco's Modified Eagle Medium at 37.degree. C. in 5% humidified CO.sub.2 incubator for a time period ranging between 15 minutes and 30 minutes; c. diluting the digested mixture from step (b) with Dental Pulp Complete Culture Media 1 (DP-CCM1) comprising DMEM-KO at 73%, fetal bovine serum at 20%, penicillin-streptomycin at 5%, and glutamax at 2% d. centrifuging the diluted mixture from step (c) to obtain a pellet; e. re-suspending the pellet from step (d) in a fresh Dental Pulp Complete Culture Media 1, and f. culturing the cells obtained from the pellet of step (e) in incubator till the confluency of the cells reaches between 80% and 85%. 3. The method as claimed in claim 2, wherein the desired size is between 0.8 mm.sup.3 and 1.5 mm.sup.3. 4. The method as claimed in claim 1, wherein the method step of sequential sub-culturing comprises steps of: a. removing the conditioned media from the preceding primary stock culture; b. rinsing the stock culture with Dulbecco's Phosphate Buffered Saline (DPBS); c. trypsinizing the stock culture with Trypsin-EDTA in an amount ranging between 0.5 ml and 2.5 ml; d. adding Dental Pulp Complete Culture Media 2 (DP-CCM2) comprising DMEM-KO at 83%, fetal bovine serum at 10%, penicillin-streptomycin at 5%, and glutamax at 2% been; e. centrifuging the resulting cell suspension at 1200 rpm for 6 minutes at room temperature (18.degree. C..+-.2.degree. C.) to obtain a pellet; f. reconstituting the cell suspension by re-suspending the pellet in fresh DP-CCM2; g. dividing the resulting cell suspension from step (f) into a quality control test sample, a retention cell culture and an expansion cell culture; h. subjecting the expansion cell culture to sub-culturing in DP-CCM2 in an amount ranging between 800 cm.sup.2 and 2500 cm.sup.2; and i. incubating the cells at 37.degree. C. in 5% humidified CO.sub.2 incubator till the confluency of the cells reaches between 80% and 85%. 5. The method as claimed in claim 1, wherein the method step of cryo-preserving comprises steps of: a. adding Cell Freezing Media to the cells from the third stock culture, wherein the Cell Freezing Media comprises DMSO at 10%, human serum albumin at 10%, penicillin-streptomycin at 5%, and glutamax at 2%; b. centrifuging the resultant mixture to obtain a pellet; c. re-suspending the pellet in a fresh Cell Freezing Media; d. sealing the reconstituted Cell Freezing Media based cell suspension in a cryobag; e. transferring the cryobag to a Controlled Rate Freezer wherein the temperature is reduced gradually at 1.degree. C. per minute till it reaches -80.degree. C.; and f. transferring the cryobag to vapour phase of liquid nitrogen storage freezer for storage purposes.

Details for Patent 9,839,654

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2034-08-20