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University Stem Cell Center

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University Stem Cell Center University Stem Cell Center Presentation Transcript

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

    Collagenase Solution
    A. Dissolved residue of
    fat and oil layer ->
    B. Collagenase solution layer->
    C. stem cell layer(white line)->
    D. RBC layer ->
    pure fat
    4-1. Mixing fat(2) with collagenase solution
    • Mix the separated pure adipose with Collagenase solution(1%) in the ratio of 1:1 or 1:0.5.
    4-2. Warm-up
    Put it into Shaking incubator at 37℃, 200 rpm for 30 minutes.
    6. The result of 5ml
    5. leave 5ml and remove A and B
    4-3. Centrifuge the mixture of collagenase solution and adipose at 800G for 5 minutes.
    Stem cell layer
    4.After centrifuge at 800G , 5 min
  • 7. Neutralization of collagenase Solution
    1. Mix the patient's serum and normal saline to neutralize Collagenase solution.
    2.Centrifuge at 300G for 3minutes
    3.Remove the outcome except 5ml using pipette.
    1.Mixing the result with Serum
    2.Mixing with Normal Saline and do hand shaking
    Serum
    • Serum and
    Normal Saline
    3. Result after 300G, 3min centrifuge
    4.Leaving bottom of 5ml, remove all
  • 8. Adipose-derived stem cell washing:
    2. hand shaking and centrifuge at 300G, 3min
    1. Mixing with Normal Saline(around 40cc)
    3.Remove the outcome except 5ml
    4.The result after Washing
    6-1. Washing using Normal Saline
    -> Mix 35ml Normal Saline and shake by hands.
    6-2. Centrifuge at 300G for 3 minutes.
    6-3. Remove the outcome (Stem cells, Normal Saline,
    RBC) except 5ml using pipette.
    6-4. Repeat these procedure (6-1~6-3) 3 times.
    7-1. Remove the final residue
    -> Filter with 100㎛ Cell Strainer to avoid tangles when inject stem cell with a needle.
    1.Result after step 6-4
    2.put 100㎛Cell Strainer in new conical tube
    3. Filter the result with 100㎛Cell Strainer
    4.Stem cells for clinical use
     100㎛Cell Strainer
  • 9. Stem cell and fat transplantation
    Stem cell and fat transplantation
    • Transfer to injection syringe
    • Inject fat into the treatment area
    using blunt tipped injection cannula.
    Two Injection Methods
    - injecting stem cell and pure fat separately.
    - injecting stem cell and pure fat together.
  • Volume of Compressed Fat 1:
    (Fat + ADSCs + PRP)
  • Volume compressed Fat 2:
    (Fat + ADSCs + PRP)
  • The grafted fat survives !
    Microscopic Photo: neo-vascularized in vivo fat graft
    • Cell count : hemacytometer
    : 1-5 x 1000000 / ml
    • Cell viability test : tryphan blue(0.4%)
    cell wall dyeingalive
    dark dyeingdead
  • Hemacyometer
    concentration(cee/ml)=
    avg # of cell x Dilution factor x 10000/sq
    # of cell/sample =concentration(cell/ml) x volume of sample
  • Hemacyometer- counting cells
    x 200
  • Hemacytometer
    - counting cells
    x 200
  • 1) Stem cell separation for clinical use -
    erythocytes present in culture
  • 1) Purified ADSCs - erythocytes have been
    removed for cell counting
    Removal of erythrocytes X 200
  • Purified ADSCs
  • Comparison
    ADSC for clinical use
    Purified ADSC
  • ADSC culture 2 days
    x 100
  • Stem cell differentiation into Fibroblasts
    Before
    A. earlyattachment
    After
    * Methods in Molecular Biology
  • Stem cell derived Fibroblast - high power
    x 200
  • ADSC culture 5days
    x 100
  • ADSC Culture (high power) - 5 days
    x 200
  • ADSC culture 10days
    x 200
  • Breast Augmentation: 125cc Fat & ADSCs per Breast
    Before
    After
  • Breast Augmentaion: 125cc Fat & ADSCs per Breast
    Before
    After
  • Before
    After 4 Weeks
  • Breast Augmentaion: 150cc Fat & ADSCs per Breast
    Before
    After
  • Breast Augmentaion: 150cc Fat & ADSCs per Breast
    Before
    After
  • Breast Augmentation with ADSCs:
    20 month follow-up
    Before
    After
  • Breast Augmentation with ADSCs:
    20 month follow-up
    After
    Before
  • Breast Augmentation with ADSCs:
    8 month follow-up
    Before
    After
  • Breast Augmentation with ADSCs:
    6 month follow-up
    Before
    After
  • Facial Grafting with ADSCs:
    After
    Before
  • Facial Grafting with ADSCs:
    After
    Before
  • Facial Grafting with ADSCs:
    After
    Before
  • Facial Grafting with ADSCs:
    Before
    After 6 month
  • Facial Grafting with ADSCs:
    Before
    After 6 month
  • Facial Grafting with ADSCs:
    Before
    After 6 month
  • Facial Grafting with ADSCs:
    Before
    After 6 month
  • Facial Grafting with ADSCs:
    Before
    After 11 month
  • University
    Stem Cell
    Center
    Conclusions
    ADSCs are
    injected back
    into the patient
    Fat cells are
    harvested via
    liposuction
    ADSCs are
    Isolated with
    the Multistation
  • Conclusions
    Adipose-derived stem
    cells can be easily
    harvested from fat
    using liposuction
  • Conclusions
    ADSCs can be safely and
    successfully isolated with
    the Multistation machine
    in the operating room
  • Conclusions
    The isolated ADSCs can
    be injected back
    into the patient on
    the same day of surgery
    without culturing the
    cells or banking the cells
  • Conclusions
    This concept/system reduces the time
    and cost of stem cell therapy by a
    factor of 10X, compared to bone
    marrow derived stem cell therapy