Closed culture system without an enzyme & Closed cell banking system

Transcription

1 Closed culture system without an enzyme & Closed cell banking system Lee, Hee-young, M.D., Ph.D. Yang, Hyun-jin, M.D., Ph.D., BaroYL plastic surgery, Medikan.Inc.

2 Final result of aging: decreased regenerating cell number Reacting cells; Effective cell numbers Result of aging; Circulating MSCs

3 Repeating, enough cell-number therapy for anti-aging; Left; de Jesus B B, and Blasco M A Circulation Research. 2012;111: Right; If the number of stem cells are maintained(blue), Normal somatic cells would be maintained(green) Disease crisis would not be occurred(red). Booster injection of cells could extend life span(purple)

4 Future; Automatic & Individualized system

5 Conventional culture have been optimized for researches; Difficult protection from contaminations Difficult processing because of passages Difficult monitoring for cell characters Difficult counting in living status Decrease of activities during transportation De-capping effect of liquid nitrogen

6 OUR CULTURE METHODS ; LISS In clinic closed scraping culture & banking system

7 Lee s Infinite Stem cell culture System ; clinical cell expansion Medical protocol; rubber port and needle Complete closed culture system; harvesting to banking Easier techniques; no washing during passages Prevention of cross contamination in LN2; water welding capsule No enzyme, no reagent; magnet blade scraping

8 Minimal clinical setting

9 Pressure controlled incubator Both + / - pressure Timer Ph, PCO2, DO monitoring Gas solubility control

10

11 Why do you use strong proteinase, like trypsin? Trypsin in principle is considered to cut the limbs of attaching cells, animal stem cells to transfer them to new cell culture dish with fresh nutrients.(detaching & dividing) Porcine (R/O swine virus) Recombinant (R/O unknown genes)

12 Trypsin- DNA damage Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells Yolanda Lorenzo1*, Kristiane Haug Berg1, Kristine Ustgaard-Andersen1, Erik Otter Johnsen1, Amund Ringvold1,2, Morten C. Moe1,2, Liv Drolsum1,2, Katerina Jirsova4, Bjørn Nicolaissen1,2 and Andrew Collins3

13 Scraping mechanisms

14 Megakit-PET; 1 million cells at cnfluency

15 Gigakit-PET; A4 size 13 million cells at confluency

16 Megakit-transport-PET; 0.3 million cells at confluency

17 Megakit-Polystyrene; 160x160 6 million cells at confluency

18 Processing time for a passage

19 Result cell migration ADSCs Media spray After 1day Cell migration

20 Scraping & Attaching Numbers instead of Passage Numbers (P0, P1, P2..).

21 Applications Culture(Day4) Cell Scraping Culture(Day4) Cell therapy Cell & Theraform TM mixing Implant 40X Direct injection

22 Continuous culture & harvesting kit Phenol free DMEM & FBS filling with syringe CO2 vacuum incubator Magnetic internal scraper Cell harvesting Image analyzing cell counter (patent pending)

23 Colony pattern inspection

24 Hemocytometor manual counting Image analyzing program counting Attached cell counting during culture

25 FREEZING AND BANKING

26 Freezing rates control system! minimize Ice crystal injury Homogeneous ices, no component separation

27 Super cooling effect decrease hyperosmolar injuries. Simultaneous ice formation within several seconds! Minimized liquid movement period. Diffusion time along density gradient is minimized. Relatively even crystal formation.! Minimize membrane movement and tearing.

28 Homogenous ice maker (Cryo-Homogenizer ) Freezing under 3 atm pressure Homogenous blending cells & cryo-protectants Super cooling - Homogenous ice crystal 1

29 AIDS could be Air born disease.

30 Perfect protection and easier picking up. Homogenous Single body No sealing structure Very stable in LN2 Sterilizer containing Water mass is expanded but plastic is contracted.

31 LN2 proof ice capsule ; Time Capsule Assamble and D.W charging Freezing in LN 2

32 Clinical Cases EVIDENCES OF REAL REGENERATIONS

33 Acute trauma; skin defect! glands, hair follicle regrowth! full thickness regeneration SVF injection Cell Cell,pre treatment 4days 3 weeks 311 months 33

34 Cx. of Lower Blepharoplasty; F/56 SVF inj. Allogenic Cell Pre Treatment Day 1 Day 3 Day 7 Day 12 Day 14 Cell Cell Day 16 Day 19 Day 36 Day36 Day

35 Embolism and full layer skin defect; M/30 Pre-Tx. 7 days 21 days 24 days 37 days 46 days 49 days 8 weeks 10 weeks 16 weeks 35

36 Progressing contracture 16 weeks 22 weeks 24 weeks 11 months 20 months 27 months 37months 49 months 55 months 57 months 36

37 Progeria (Hutchinson-Gilford syndrome) pathology LMNA gene mutation Lamin A- one misspelling. Nucleus crumpling! cell division problem like aging Decreased stem Cell number symptoms Normal at birth 8-10 time faster than normal persons after 9~18months after birth. Different with adult progeria(werner syndrome) Aged skin, decreased body fat and hair, prominent veins, nail crumpling, joint stiffness Microgenia, exophthalmos, low stature atherosclerosis, cardiac problem, CVA Average life span; 13 years

38 H-G progeria; scalp thickening, hair growth, stature increase, weight gain Pre Treatment 9-year-old boy 7 Months

39 Pre Treatment 7 Months 39

40 Nail crumpling 1 month after Tx. 4 months after Tx. 40

41 Joint stiffness of hands, LOM 3 months after Tx. 7 months after Tx. 41

42 stature 7 months after Tx year after Tx

43 16 years, 650 patients, 2,161 cases, 3.3times/ 1patient Cancer occurrence; 0%

44 16 years, 650 patients, 2,161 cases; No safety problem, valid effects complication; 0.79% I.V.; 13/1410(0.85%) Local; 4/748(0.41%) Intrathecal; 0/3(0.00%) caner; 0% 0/650, 2161 injection for 16 years Satisfaction rate excluding Placebo effects; 65~70%

45 Conclusions Many stem cell therapies need repeated injection. Total injected cell number is very important, so more efficient, easier, cheaper culture system is necessary. Banking contamination could be a trouble. MSCs culture is easier than fish breeding. No malignant transformation, nothing to loose.

46 Thank you very much! Now, doctors can keep their health, and can extend their life span. Why not?