Claims for Patent: 10,479,978

Summary for Patent: 10,479,978

Title:	Postnatal adherent cells and preparation method therefor
Abstract:	Enhanced postnatal adherent cells and a preparation method therefor are provided. The preparation method of enhanced postnatal adherent cells can increase the yield and the proliferation rate of adherent cells from placental tissues; and prepare adherent cells, which secrete proteins effective for neurological diseases and have an improved ability for movement to damaged tissues.
Inventor(s):	Kim; Hye Sun (Seongnam-si, KR), Kang; Ah Reum (Seongnam-si, KR), Kim; Hyun Ju (Seongnam-si, KR), Park; Kung Mi (Seoul, KR), Joo; Sang Un (Seoul, KR)
Assignee:	CHA BIOTECH CO., LTD. (Seoul, KR)
Application Number:	15/577,633
Patent Claims:	1. A method of preparing enhanced postnatal adherent cells, the method comprising: obtaining an amniotic tissue from a detached placenta; harvesting cell populations by adding an enzyme mixed solution to the amniotic tissue, wherein the enzyme mixed solution comprises collagenase at a concentration of 0.5 mg/ml to 5 mg/ml, trypsin at a concentration of 1 mg/ml to 5 mg/ml, DNA hydrolase (DNase) at a concentration of 0.01 mg/ml to 0.05 mg/ml, and dispase at a concentration of 0.1 U/ml to 5 U/ml; isolating the enhanced postnatal adherent cells by culturing the harvested cell populations in a container by adhesion culture and then treating the cell populations with an animal component-free (ACF) recombinant enzyme; and subculturing the isolated enhanced postnatal adherent cells under a hypoxia condition lower than a normoxia condition of 21% in a medium containing fibroblast growth factor-4 (FGF-4) and heparin at least 6 passages, wherein the subculturing further comprises treatment of the animal component-free recombinant enzyme before transferring the cells for subculturing at every stage of subculturing, and wherein 80% or more of the adherent cells express CD44, CD73, CD90, and CD105-positive surface markers and 10% or less thereof express a CD45-negative surface marker, and more vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) are secreted compared to the isolated enhanced postnatal adherent cells of passages 1 to 4. 2. The method of claim 1, wherein the amniotic tissue from the amniotic tissue is obtained by scraping a chorionic plate membrane of the placenta to remove a chorion. 3. The method of claim 1, wherein the enzymatic reaction is performed at 30.degree. C. to 40.degree. C. for 5 minutes to 60 minutes under shaking. 4. The method of claim 1, wherein the hypoxic condition is at an oxygen partial pressure of 1% to 12%. 5. The method of claim 1, wherein the subculturing is performed from passage 6 to passage 20. 6. The method of claim 1, wherein the harvesting of the cell populations increases yield of adherent cells, as compared with no use of the enzyme mixed solution. 7. The method of claim 1, wherein a proliferation rate of the adherent cells is increased, as compared with that under a normoxia condition. 8. The method of claim 1, wherein the isolating of the adherent cells increases purity of the adherent cells, as compared with no use of the animal component-free recombinant enzyme. 9. The method of claim 1, wherein the prepared adherent cells secrete a protein selected from the group consisting of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-.beta.1, hepatocyte growth factor (HGF), interleukin-6 (IL-6), progranulin, and combinations thereof. 10. A method of increasing a preparation efficiency of enhanced postnatal adherent cells, the method comprising: harvesting cell populations by adding an enzyme mixed solution to an amniotic tissue, wherein the enzyme mixed solution comprises collagenase at a concentration of 0.5 mg/ml to 5 mg/ml, trypsin at a concentration of 1 mg/ml to 5 mg/ml, DNA hydrolase (DNase) at a concentration of 0.01 mg/ml to 0.05 mg/ml, and dispase at a concentration of 0.1 U/ml to 5 U/ml; isolating enhanced postnatal adherent cells by culturing the harvested cell populations in a flask by adhesion culture and then treating the cell populations with an animal component-free (ACF) recombinant enzyme; and subculturing the isolated enhanced postnatal adherent cells under a hypoxia condition lower than a normoxia condition of 21% in a medium containing fibroblast growth factor-4 (FGF-4) and heparin at least 6 passages, wherein the subculturing further comprises treatment of the animal component-free recombinant enzyme before transferring the cells for subculturing at every stage of subculturing, and wherein 80% or more of the adherent cells express CD44, CD73, CD90, and CD105-positive surface markers and 10% or less thereof express a CD45-negative surface marker, and more vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) are secreted compared to the isolated enhanced postnatal adherent cells of passages 1 to 4. 11. The method of claim 10, wherein the increasing of the preparation efficiency of the adherent cells is increasing of yield of adherent cells, as compared with no use of the enzyme mixed solution, increasing of a proliferation rate of the adherent cells, as compared with that under a normoxia condition, or increasing of purity of the adherent cells, as compared with no use of the animal component-free recombinant enzyme.

Details for Patent 10,479,978

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2035-05-28