University Stem Cell Center

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University Stem Cell Center

  1. 1. University <br />Stem Cell <br />Center<br />“Bringing Adipose-derived <br /> Stem Cells into the <br /> Operating Room” <br />
  2. 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. 3. Embryonic Stem Cells<br /><ul><li>Mostpleuripotent, immortal in culture
  4. 4. Controversial, difficult to obtain
  5. 5. Must culture for many cell </li></ul> divisions to obtain adequate <br /> cell numbers<br />
  6. 6. Adult Stem Cells <br />Found in many different sites<br /> in the body (bone, muscle, etc.)<br />
  7. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 17. The Multistation - A high tech <br />ADSC Extraction facility<br />
  18. 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. 19. Airborne particle monitoring
  20. 20. HEPA air filtering
  21. 21. UV light sterilization
  22. 22. Angled rotor centrifuge (for</li></ul> uniform sedimentation <br /> layers of cells/PRP<br />
  23. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 34. ADSC<br />Separation<br /> Protocol<br />using the<br />MultiStation<br />System<br />
  35. 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. 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. 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. 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. 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. 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. 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. 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. 43. 9. Stem cell and fat transplantation<br />Stem cell and fat transplantation<br /><ul><li>Transfer to injection syringe
  44. 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. 45. Volume of Compressed Fat 1:<br />(Fat + ADSCs + PRP)<br />
  46. 46. Volume compressed Fat 2:<br />(Fat + ADSCs + PRP)<br />
  47. 47. The grafted fat survives !<br />Microscopic Photo: neo-vascularized in vivo fat graft <br />
  48. 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. 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. 50. Hemacyometer- counting cells<br />x 200 <br />
  51. 51. Hemacytometer<br />- counting cells<br />x 200 <br />
  52. 52. 1) Stem cell separation for clinical use - <br />erythocytes present in culture<br />
  53. 53. 1) Purified ADSCs - erythocytes have been <br /> removed for cell counting<br />Removal of erythrocytes X 200<br />
  54. 54. Purified ADSCs<br />
  55. 55. Comparison<br />ADSC for clinical use<br />Purified ADSC<br />
  56. 56. ADSC culture 2 days<br />x 100<br />
  57. 57. Stem cell differentiation into Fibroblasts <br />Before<br />A. earlyattachment<br />After<br />* Methods in Molecular Biology<br />
  58. 58. Stem cell derived Fibroblast - high power<br />x 200<br />
  59. 59. ADSC culture 5days<br />x 100<br />
  60. 60. ADSC Culture (high power) - 5 days<br />x 200 <br />
  61. 61. ADSC culture 10days<br />x 200 <br />
  62. 62.
  63. 63. Breast Augmentation: 125cc Fat & ADSCs per Breast<br />Before<br />After<br />
  64. 64. Breast Augmentaion: 125cc Fat & ADSCs per Breast<br />Before<br />After<br />
  65. 65. Before<br />After 4 Weeks<br />
  66. 66. Breast Augmentaion: 150cc Fat & ADSCs per Breast<br />Before<br />After<br />
  67. 67. Breast Augmentaion: 150cc Fat & ADSCs per Breast<br />Before<br />After<br />
  68. 68. Breast Augmentation with ADSCs:<br /> 20 month follow-up<br />Before<br />After<br />
  69. 69. Breast Augmentation with ADSCs:<br /> 20 month follow-up<br />After<br />Before<br />
  70. 70. Breast Augmentation with ADSCs:<br /> 8 month follow-up<br />Before<br />After<br />
  71. 71. Breast Augmentation with ADSCs:<br /> 6 month follow-up<br />Before<br />After<br />
  72. 72. Facial Grafting with ADSCs:<br />After<br />Before<br />
  73. 73. Facial Grafting with ADSCs:<br />After<br />Before<br />
  74. 74. Facial Grafting with ADSCs:<br />After<br />Before<br />
  75. 75. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  76. 76. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  77. 77. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  78. 78. Facial Grafting with ADSCs:<br />Before<br />After 6 month<br />
  79. 79. Facial Grafting with ADSCs:<br />Before<br />After 11 month<br />
  80. 80. 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 />
  81. 81. Conclusions<br />Adipose-derived stem <br /> cells can be easily<br /> harvested from fat <br /> using liposuction<br />
  82. 82. Conclusions<br /> ADSCs can be safely and<br /> successfully isolated with<br /> the Multistation machine<br /> in the operating room<br />
  83. 83. 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 />
  84. 84. 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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