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University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
University Stem Cell Center
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University Stem Cell Center

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  1. University <br />Stem Cell <br />Center<br />“Bringing Adipose-derived <br /> Stem Cells into the <br /> Operating Room” <br />
  2. What are stem cells? <br /> Why are so useful? <br /> they so useful ?<br />Stem Cells have the <br />potential to repair <br />or regenerate<br />damaged cells <br />(in disease) or <br />replace degenerating <br />cells (in aging)<br />Cultured Stem cells <br />
  3. Embryonic Stem Cells<br /><ul><li>Mostpleuripotent, immortal in culture
  4. Controversial, difficult to obtain
  5. Must culture for many cell </li></ul> divisions to obtain adequate <br /> cell numbers<br />
  6. Adult Stem Cells <br />Found in many different sites<br /> in the body (bone, muscle, etc.)<br />
  7. What are Adipose-Derived Stem Cells (ADSCs)? <br />They are adult<br />stem cells found<br />in ordinary fat<br />ADSCs ready for clinical use<br />
  8. Autologous Adipose <br /> Tissue-Derived <br /> Stem Cells (ADSCs)<br />Facts about ADSCs:<br /><ul><li> They are abundant - 1% of fat is composed of adult stem cells called </li></ul> “adipose derived stem cells” (ADSCs)<br /><ul><li> They are powerful - ADSCs have pleuripotent potential to grow and </li></ul> differentiate into many different types of cells<br /><ul><li> Easy to harvest - They can harvested under local anesthesia </li></ul> in the OR with liposuction techniques<br /><ul><li>Can be cultured - ADSCs can be cultured to increase cell number</li></ul> exponentially to reach cell counts of 108 cells<br /><ul><li>Can be stored - ADSCs can be frozen and banked in tissue </li></ul> banks for future use or repeated use<br />
  9. Autologous Adipose<br /> Tissue-Derived <br /> Stem Cells (ADSCs)<br />Advantages of ADSCs<br /><ul><li>Advantage over bone marrow stem cells- ADSCs can be </li></ul> used “fresh” and injected in the OR on the same day of <br /> harvest. Bone marrow stem cells have to be cultured to <br /> get adequate cell numbers. <br /><ul><li>Advantage over fat grafting - ADSCs have a much higher</li></ul> survival rate than traditional fat grafting, producing more<br /> reliable volumes for soft tissue augmentation<br /><ul><li>Advantages over embryonic stem cells - ADSC therapy</li></ul>avoids the political, ethical, and logistical clinical problems <br /> with embryonic stem cells. <br />
  10. Different Ways of Using ADSCs<br />Fresh isolated ADSCs - OK to use clinically in OR now<br /> - No FDA restrictions<br /> - treated as fat grafting<br />Culturing ADSCs – restricted to investigational use by FDA<br /> - Major FDA restrictions on clinical use<br /> - Treated as a “drug” by the FDA<br />Differentiated ADSCs- restricted to investigational use<br /> - can be differentiated into fat, <br /> bone, cartilage, blood vessels etc.<br />
  11. ADSC-enriched Fat Transplantation<br />ADSCs can be used with fat transplants to reduce fat resorption<br /><ul><li>Fat grafting is a useful technique in plastic surgery, but</li></ul> suffers from low fat cell survival rate => graft resorption<br />- ADSCs can be mixed with fat for cosmetic and reconstructive<br /> plastic surgery applications, such as breast reconstruction <br />Fat transplant survival rate increases from <br />30% to > 70%<br />Yoshimura et al., (2008) “Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells”, 49p<br />
  12. Clinical Applications of ADSCs that can be done today at University Stem Cell Center<br />Wound healing - direct injection into the wound edge<br /> - No FDA restrictions on this, provided it is done<br /> on the day of ADSC harvest<br /> - can be combined with conventional wound<br /> care treatments - Ex: non-surgical, surgical<br /> debridement, skin grafting, HBO, etc. <br />Breast surgery – breast reconstruction and cosmetic breast<br /> surgery with ADSC enriched fat grafts<br /> - Over 600 cases done in Japan with long<br /> term good results. <br />
  13. What makes stem cells<br />differentiate into specialized cells? <br />Inductive<br />Factors<br />Added to the growth<br />Medium<br />Local cell-cell communication in tissue<br />ADSCs are undifferentiated <br />
  14. Inductive Factors that make stem <br />cells differentiate into specialized cells: <br />1) Adipocyte<br />Dexamethasone,isobutylmethylxanthine, indomethacin, insulin, thiazolidinedione<br />2) Cardiomyocyte<br />Transferrin, IL-3, IL-6, VEGF<br />3) Chondrocyte<br />Ascorbic acid, bone morphogenetic protein 6, dexamethasone, insulin, transforming growth factor<br />4) Endothelial<br />Proprietary medium (EGM-2-MV; Cambrex) containing ascorbate, epidermal growth factor, basic fibroblast growth factor, hydrocortisone<br />5) Myocyte<br />Dexamethasone, horse serum <br />6) Neuronal-like<br />Butylated hydroxyanisole, valproic acid, insulin<br />7) Osteoblast<br />Ascorbic acid, bone morphogenetic protein 2, dexamethasone, 1,25 dihydroxy vitamin D3<br />
  15. University <br />Stem Cell <br />Center<br />A state-of-the-art adipose derived <br /> stem cell extraction facility<br />Purpose: To create an OR with the capability of harvesting fat,<br /> extracting the stem cells, and re-infusing them under sterile <br /> operating room conditions all in one day<br />
  16. How the Stem Cell <br /> Center Works<br />ADSCs are<br /> Isolated with<br />the Multistation<br />ADSCs are<br />injected back<br />into the patient<br />Adipose tissue <br />Harvested with <br />liposuction<br />
  17. The Multistation - A high tech <br />ADSC Extraction facility<br />
  18. Multistation<br />Features<br /><ul><li> Controlable acceleration </li></ul> and deceleration rates<br /><ul><li> Shaking cell incubator </li></ul> (for collagenase bath)<br /><ul><li> Can centrifuge blood and fat </li></ul> (simultaneously)<br /><ul><li> Can be used for PRP isolation </li></ul> or for ADSC extraction<br /><ul><li>“Clean bench” technology
  19. Airborne particle monitoring
  20. HEPA air filtering
  21. UV light sterilization
  22. Angled rotor centrifuge (for</li></ul> uniform sedimentation <br /> layers of cells/PRP<br />
  23. Multi Station Features<br />1 . Clean Bench - type III air flow<br />Type III: Biohazard Type <br /> (Multi Station)<br />Type I: HorizontalType<br />Type II: Vertical Type<br />Air is absorbed and <br />circulates from the frontand back sides by the vertically circulating air current method, and Air is released through the upper outlet.<br />A filter is equipped inside by the horizontal air current method. Air is released to the operator’s front.<br />A filter is equipped inside by the vertical air current method. <br />Air is released to the operator’s front.<br />
  24. Multi Station Features<br />2 . Particle Meter<br />Special features of Particle Meter<br />1. Detects the Hygienic Statusof machine`s inside for operation.<br />2. Gives the operator a visual indicator in order to make prompt judgments during cell processing. <br />3. Confirms current status on a constant basis, and measures the time to replace the HEPA FILTER by indicating its contamination rate.<br />Patent Description<br />Application No.20-2006-0032746 (2006.12.28) <br />Registration No. 20-0438027-0000 (2008.01.08)<br />
  25. Multi Station Features<br />3 . HEPA Filter<br />- Incorporated to act as a Dust Collector; it eliminates 99.98% of<br /> 0.03µm-sized airborne particles, which is 1/3 of the size of smoke <br /> particles.<br />- Maintains the perfect hygienic status by integrating HEPA Filter <br /> into Multi station.<br />
  26. 4 . UV Light<br />Multi Station Features<br />Duplex 254nm germicidal UV light designed for <br />Sterilization. (inside of Clean Bench and above <br />HEPA Filter)<br />
  27. Multi Station Features<br />5 . Centrifuge<br />Types of Rotors<br /> Swing Rotor <br />(Multi Station)<br />Angle Rotor<br />(traditional centrifuge)<br /> Fat/fluid<br /> sedimentation<br /> layering with<br /> the two different<br /> types of rotors:<br />
  28. Multi Station Features<br />5 . Centrifuge<br />Automatic Calculation of RCFMicroprocessor controlled, developed exclusively for the centrifuge to operate<br />in 3 modes. RPM and RCF factors can<br />be used for adjusting revolution rate.<br />10 Levels of Acceleration/Deceleration<br />No sudden acceleration or sudden braking,<br />Accelaration rate and slow deceleration is completely adjustable for sample separation<br />Reliable Safety <br />Several safety devices incorporated into design, including self-diagnose function, sensor device for disproportionate rotor speed, etc.<br />Simple InstructionEasy to operation by touch sensing LCD screen with interactive menu, built-in memory of the 200 most frequently-used-settings.<br />Interchangeable Slots (buckets)<br /> slots can be used for either<br /> 15cc vials (PRP) or 50cc <br /> buckets (for fat).<br />Brushless D.C.Motorno need for replacing motor<br /> brushes, no carbon powders <br /> released, no maintenance <br /> expenses required.<br />
  29. Multi Station Features<br />6 . Shaking incubator<br />Accurate Temperature Regulationwith digital microprocessor control<br />Auto-Stoppingfeature stops shaking motion when the door opens during operation.<br />Auto-Restartsafter electricity failure or when the door is shut.<br />Uniform Temperature Accuracywith built-in air circulation system <br />Vibration & Noise freedue to plate type brushless DC motor<br />Operating Status visible from outsidewith transparent acrylic door<br />
  30. Multi Station Features<br />7 . Accessories<br />Pipette<br /> ”ISO 9001 certified”, Autoclavable (121℃, 20min.)<br />Conical Centrifuge Tubes - for blood and fat <br />“ ISO 9001 certified” <br />“ FDA certified” <br />“ Gamma sterilization mark attached”<br />
  31. Multi Station Features<br />7 . Accessories<br />Pipette Tips<br />“ISO 9001 certified” <br />“FDA certified” <br />“Gamma sterilization mark attached”<br />Cell Strainer <br />“Gamma sterilization mark”<br />
  32. Multi Station vs Celution vs Lipokit<br />Celution 800/CRS System <br /> (Cytori Corp.)<br />Multi Station <br />(P&C International Corp.)<br />Lipo-Kit (Medikan Corp.)<br />
  33. Multi Station vs Celution vs Lipokit<br />Fat Volume that can be processed<br />Machine Manufacturer<br />Type of Machine<br />300cc maximum fat volume/run<br />Cellution System (Cytori Corp.)<br />Automated fat processing<br />No limit to fat volume/run<br />Multistation (P&C Corp.)<br />Manual fat processing<br />50cc maximum fat volume/run<br />No fat processing (just a centrifuge)<br />Lipo-Kit (Medikhan Corp.)<br />Automated fat processing<br />Unknown fat volume/run<br />YC-100<br />(Medikhan Corp.)<br />
  34. ADSC<br />Separation<br /> Protocol<br />using the<br />MultiStation<br />System<br />
  35. 1. Blood/serum acquisition<br />ADSCs Separation Protocol <br />Using Multi Station<br />Extracting blood<br />1. Extract blood from a patient to acquire serum. <br /> *The patient’s serum is necessary to neutralize <br /> Collagenase Solution (for step 3) <br /> *Amount of blood needed: 6~10cc blood for <br /> pure fat 20-30ml injection<br /> 1000G3min<br />2. Centrifuge the 6~10cc blood at <br /> 1000G for 3minutes.<br />Result after centrifuge<br />3. You can acquire 3cc~5cc of serum (from <br /> 6~10cc blood). Store the serum at Shaking Incubator at 37˚C and 150 RPM<br />Serum: Yellow part<br />
  36. 2. Fat acquisition<br />ADSCs Separation Protocol <br />Using Multi Station<br /> 1. Liposuction planning<br /> - Decide on where to perform liposuction and fat injection.<br /> - Tool : Marker pen<br /> 2. Tumescent solution infiltration<br /> - Contents of tumescent solutionvolume<br /> 0.9% NaCl or H/S 1000ml<br /> 2% Lidocaine 600mg(=30ml)<br /> 1:1000 Epinephrine 1ml<br /> 8.4% Sodium bicarbonate(NaHCO3) 10ml<br /> Triamcinolone 10mg(=1/4 ample)<br /> ※ Inject Tumescent solution into the treatment area in the <br /> ratio of 1:1 or 1:2 using Cannula. (Tumescent:Fat to be injected)<br /> 3. Standby before liposuction<br /> - After injecting Tumescent solution, standby for 30~40minutes forthe <br />effect of anesthesia to occur.<br />
  37. ADSCs Separation Protocol <br />Using Multi Station<br />3. Liposuction<br />2. Transferring lipoaspirate <br /> into conical tubes<br />1.Manual liposuction<br />- Extract fat using Liposuction Cannula after combining the Luer-Lock with an injector.<br />※ Areas that contain the least fiber: thigh -> abdomen -> waist <br /> - Tools : Injector(20cc, 50cc), Luer-lock(20cc, 50cc), Lipo Suction Cannula<br />Fat(2) For stem cell separation<br />Fat(1) For Injection<br />3. Lipoaspirate in conical tubes<br />4. Zoomed-in lipoaspirates<br />
  38. 4. Setting aside Fat For Injection<br />(Pure fat will be mixed with stem cell before injection)<br />1. Put the conical tube into Centrifuge within 1000G for 3~5minutes.<br />2. After centrifuge<br />1.1000G, 3~5minutes<br /> Fat layer<br /> 2. Put the pipette deeply into the inside of conical tube and extract A part<br /> (free oil, tumescent solution, and the RBC).<br />Free oil & Tumescent & RBC layer<br />4. Pure fat without free oil <br /> tumescent fluid and RBC<br />3. Remove Free oil & Tumescent fluid & RBC layer (A part)<br />After removing free oil(upper part) remove tumescent and RBC (low part)<br />A<br />
  39. 5. Preparing Fat For stem cell transplantation:<br /> 1. Put the conical tube into Centrifuge at 100G for 3minutes.<br />2. After centrifuge<br />1.100G, 3minutes<br />2. Put the pipette deeply into the inside of conical tube.<br />3. Extract A part(free oil, tumescent solution, and the RBC).<br /> Fat layer<br />Free oil & Tumescent & RBC layer<br />4. Pure fat without free oil tumescent and RBC<br />3. Remove Free oil & Tumescent & RBC layer (A part)<br />After removing free oil(upper part) remove tumescent and RBC (low part)<br />A<br />
  40. 6. Cell separation with collagenase processing:<br />2.After hand shaking, put it in incubator at 37℃,200 rpm, 30 minutes.<br />1.Mixing Collagenase Solution with pure fat<br />3. Fat after Shaking incubation<br /><br />Collagenase Solution <br />A. Dissolved residue of <br /> fat and oil layer -><br />B. Collagenase solution layer-><br />C. stem cell layer(white line)-><br /> D. RBC layer -><br />pure fat<br />4-1. Mixing fat(2) with collagenase solution<br /><ul><li> Mix the separated pure adipose with Collagenase solution(1%) in the ratio of 1:1 or 1:0.5.</li></ul>4-2. Warm-up<br />Put it into Shaking incubator at 37℃, 200 rpm for 30 minutes.<br />6. The result of 5ml<br />5. leave 5ml and remove A and B<br />4-3. Centrifuge the mixture of collagenase solution and adipose at 800G for 5 minutes. <br />Stem cell layer<br />4.After centrifuge at 800G , 5 min<br />
  41. 7. Neutralization of collagenase Solution<br />1. Mix the patient's serum and normal saline to neutralize Collagenase solution.<br />2.Centrifuge at 300G for 3minutes<br />3.Remove the outcome except 5ml using pipette. <br />1.Mixing the result with Serum <br />2.Mixing with Normal Saline and do hand shaking<br />Serum<br /><ul><li>Serum and</li></ul>Normal Saline<br />3. Result after 300G, 3min centrifuge<br />4.Leaving bottom of 5ml, remove all<br />
  42. 8. Adipose-derived stem cell washing:<br />2. hand shaking and centrifuge at 300G, 3min<br />1. Mixing with Normal Saline(around 40cc)<br />3.Remove the outcome except 5ml<br />4.The result after Washing<br />6-1. Washing using Normal Saline<br />-> Mix 35ml Normal Saline and shake by hands.<br />6-2. Centrifuge at 300G for 3 minutes.<br />6-3. Remove the outcome (Stem cells, Normal Saline, <br /> RBC) except 5ml using pipette. <br />6-4. Repeat these procedure (6-1~6-3) 3 times.<br />7-1. Remove the final residue<br />-> Filter with 100㎛ Cell Strainer to avoid tangles when inject stem cell with a needle.<br />1.Result after step 6-4<br />2.put 100㎛Cell Strainer in new conical tube<br />3. Filter the result with 100㎛Cell Strainer<br />4.Stem cells for clinical use<br /> 100㎛Cell Strainer <br />
  43. 9. Stem cell and fat transplantation<br />Stem cell and fat transplantation<br /><ul><li>Transfer to injection syringe
  44. Inject fat into the treatment area </li></ul> using blunt tipped injection cannula.<br />Two Injection Methods<br /> - injecting stem cell and pure fat separately.<br /> - injecting stem cell and pure fat together.<br />
  45. Volume of Compressed Fat 1:<br />(Fat + ADSCs + PRP)<br />
  46. Volume compressed Fat 2:<br />(Fat + ADSCs + PRP)<br />
  47. The grafted fat survives !<br />Microscopic Photo: neo-vascularized in vivo fat graft <br />
  48. <ul><li>Cell count : hemacytometer</li></ul> : 1-5 x 1000000 / ml<br /><ul><li>Cell viability test : tryphan blue(0.4%)</li></ul> cell wall dyeingalive<br /> dark dyeingdead<br />
  49. Hemacyometer<br />concentration(cee/ml)= <br /> avg # of cell x Dilution factor x 10000/sq<br /> # of cell/sample =concentration(cell/ml) x volume of sample<br />
  50. Hemacyometer- counting cells<br />x 200 <br />
  51. Hemacytometer<br />- counting cells<br />x 200 <br />
  52. 1) Stem cell separation for clinical use - <br />erythocytes present in culture<br />
  53. 1) Purified ADSCs - erythocytes have been <br /> removed for cell counting<br />Removal of erythrocytes X 200<br />
  54. Purified ADSCs<br />
  55. Comparison<br />ADSC for clinical use<br />Purified ADSC<br />
  56. ADSC culture 2 days<br />x 100<br />
  57. Stem cell differentiation into Fibroblasts <br />Before<br />A. earlyattachment<br />After<br />* Methods in Molecular Biology<br />
  58. Stem cell derived Fibroblast - high power<br />x 200<br />
  59. ADSC culture 5days<br />x 100<br />
  60. ADSC Culture (high power) - 5 days<br />x 200 <br />
  61. ADSC culture 10days<br />x 200 <br />
  62. Breast Augmentation: 125cc Fat & ADSCs per Breast<br />Before<br />After<br />
  63. Breast Augmentaion: 125cc Fat & ADSCs per Breast<br />Before<br />After<br />
  64. Before<br />After 4 Weeks<br />
  65. Breast Augmentaion: 150cc Fat & ADSCs per Breast<br />Before<br />After<br />
  66. Breast Augmentaion: 150cc Fat & ADSCs per Breast<br />Before<br />After<br />
  67. Breast Augmentation with ADSCs:<br /> 20 month follow-up<br />Before<br />After<br />
  68. Breast Augmentation with ADSCs:<br /> 20 month follow-up<br />After<br />Before<br />
  69. Breast Augmentation with ADSCs:<br /> 8 month follow-up<br />Before<br />After<br />
  70. Breast Augmentation with ADSCs:<br /> 6 month follow-up<br />Before<br />After<br />
  71. Facial Grafting with ADSCs:<br />After<br />Before<br />
  72. Facial Grafting with ADSCs:<br />After<br />Before<br />
  73. Facial Grafting with ADSCs:<br />After<br />Before<br />
  74. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  75. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  76. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  77. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  78. Facial Grafting with ADSCs:<br />Before<br />After 11 month<br />
  79. University<br />Stem Cell<br />Center<br /> Conclusions<br />ADSCs are<br />injected back<br />into the patient<br />Fat cells are<br />harvested via<br />liposuction<br />ADSCs are<br /> Isolated with<br />the Multistation<br />
  80. Conclusions<br />Adipose-derived stem <br /> cells can be easily<br /> harvested from fat <br /> using liposuction<br />
  81. Conclusions<br /> ADSCs can be safely and<br /> successfully isolated with<br /> the Multistation machine<br /> in the operating room<br />
  82. Conclusions<br />The isolated ADSCs can<br /> be injected back<br /> into the patient on<br /> the same day of surgery<br /> without culturing the<br /> cells or banking the cells<br />
  83. Conclusions<br />This concept/system reduces the time<br /> and cost of stem cell therapy by a <br /> factor of 10X, compared to bone <br /> marrow derived stem cell therapy<br />

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