Automated Extraction & Culture System of Adipose …doc.xueqiu.com/144d468295a2f23f9789fc75.pdf · Automated Extraction & Culture System of Adipose-derived Stem Cells ... Potential

01. 01. Bio MasterBio Master의의 개요개요

Automated Extraction & Culture Automated Extraction & Culture System of AdiposeSystem of Adipose--derived Stem Cellsderived Stem Cells

2008.2.242008.2.24--revised revised

Dr. H.Y.Lee, Dr.H.J.Yang, Dr. H.Y.Lee, Dr.H.J.Yang, KangNamKangNam Plastic Surgery Clinic, Plastic Surgery Clinic, MediMedi--Khan(USA) Inc.Khan(USA) Inc.


Research & Trend of Stem Cell

01. 01. Bio MasterBio Master의의 개요개요Importance in Research of Adult Stem Cell

Can forecast it as Faster Growth IndustryCan forecast it as Faster Growth IndustryExpected to grow by 18.5 % to 10.9 Billion Expected to grow by 18.5 % to 10.9 Billion US Dollars by 2015.US Dollars by 2015.(Jain (Jain PharmaBiotechPharmaBiotech Report, 2005)Report, 2005)

Can be Utilized as PatientCan be Utilized as Patient--specific Stem Cell specific Stem Cell for Cell Therapyfor Cell Therapy

Over 101 millions ofOver 101 millions of worldworld--wide patients Needwide patients NeedCell Therapy. (Perry D., Science 287, 1423, 2000)Cell Therapy. (Perry D., Science 287, 1423, 2000)

Superiority in Technological CompetitivenessSuperiority in Technological Competitiveness

Current AdiposeCurrent Adipose--derived Stem Cell Researchderived Stem Cell ResearchStays at Early Stage. So, Easier to possess Stays at Early Stage. So, Easier to possess Technological Superiority than other Research.Technological Superiority than other Research.

Can Obtain Large Quantity o f Stem CellsCan Obtain Large Quantity o f Stem Cells

Diversified Technologies in IsolatingDiversified Technologies in Isolating& Culturing Adult Stem Cells& Culturing Adult Stem Cells

Can Overcome Issues over Embryonic Stem Cell ResearchCan Overcome Issues over Embryonic Stem Cell ResearchEthical Issue Ethical Issue

Difficulty in Regulating DifferentiationDifficulty in Regulating DifferentiationRequired Long Time before Commercial UseRequired Long Time before Commercial Use

Can Isolate Stem Cells from Fat Tissues Can Isolate Stem Cells from Fat Tissues

Lots of adult stem cells exist in fat tissuesLots of adult stem cells exist in fat tissuesAdult Stem Cell

01. 01. Bio MasterBio Master의의 개요개요Potential Applications of Adipose-derived Stem Cells

Wound Healing

Plastic Surgery

Bone & Cartilage regeneration

The Major advantages of adipose tissues as a source of regenerative cells• High Yield : A therapeutic dose of regenerative cells can be isolated within

about 1~2 hours without cell culture.

Safety : Patients receive their own cells (autologous-use), so there is no riskof immune rejection or disease transmission.

Versatility : Stem cells from adipose tissues can impart benefits from multiple mechanisms-of-action as pluripotent progenitor cells.

Spees JL, Olson SD, Ylostalo J, et al.: Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA.100:2397-2402, 2003

Neuron & Heart regeneration Safford, K.M., Hicok, K.C., Safford, S.D., Halvorsen, Y.D., Wilkison, W.O., Gimble, J.M., and Rice,

H.E.Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem. Biophys. Res. Commun.294, 371, 2002.

Planat-Benard, V., Menard, C., Andre, M., Puceat, M., Perez, A., Garcia-Verdugo, J.M., Penicaud, L., and Casteilla : L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ. Res. 94, 223, 2004.

Kotaro Yoshimura, Katsujiro Sato, Noriyuki Aoi, Masakazu Kurita, Toshitsugu Hirohi and Kiyonori Harii : Cell-assisted lipotransfer (CAL): supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Aesthetic Plastic Surgery, 1432-5241, 2007

Dragoo, J.L., Samimi, B., Zhu, M., Hame, S.L., Thomas, B.J., Lieberman, J.R., Hedrick, M.H., and Benhaim : P.Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads.J. Bone Joint. Surg. Br. 85, 740, 2003.


Necessity in Technological Development

01. 01. Bio MasterBio Master의의 개요개요Necessity in Technological Development I

Required Massive Cell Isolation &

Culture Technology

Required Massive Cell Isolation &

Culture Technology

Required to develop Effective

Cell Isolation Technology

Required to develop Effective

Cell Isolation Technology

1

2

3

Increased Need for

AutologousImplantation

Increased Need for

AutologousImplantation

Autologous Fat or Stem Cell Implantation for Cosmetic or plastic SurgeryAutogenous Stem cell Implantation for Burnt and Depressed Body Parts and Nerve Regeneration

Harvesting Fat Tissues → Isolation of Stem Cells → Massive Culturing →Implantationto Patients (Autologous use)

in vivo system : Employing the optimized environment for stem cell isolation. ( Can maintain constant temperature and humidity and prevent contamination)

Reduced time of stem cell isolation, Saved Cost, Least LaborWith optimized technology and automated isolation system, quantified data is available.Designed to be suitable for GMP facility or for production system of stem cells for celltherapy.

CollagenaseTreatment

For 1~2 hrs Isolation after centrifugation, oil removal and washing processes

Culturing for 10~20 days

Implantation

It takes 2~3 hours to isolate stem cells by manual methods. Faster process is required.The existing isolation technologies require many experts with much labor cost.To utilize stem cells for treatment purpose, quantified data are needed.

01. 01. Bio MasterBio Master의의 개요개요Necessity in Technological Development II

Harvest of highly

concentrated fat tissues

Isolation of Adipose-derived Stem Cells

Cell BankingIncreased need for organ storage for autologous

implantation.

Trial of homogenous(Heterogeneous) implantation

- Trial of cell therapy for brain, spine and heart with

low immunorejection

- Cell banking system is required for massively

extracted and stored stem cells for cell therapy.

Cell Therapy

After massively culturing adipose-derived stem cells, differentiation of ASCsinto adypocytes, osteocytes or neurocytes for required treatment purpose.

Implantation into patients

Cosmetic Surgery

Transplantation of fat cells and ACSs for cosmetic surgeries like breast augmentation, facial microinjection and wrinkle removal

Showing good surgery results from ASC implantation.


Current Trends in Technological Development

01. 01. Bio MasterBio Master의의 개요개요Current Trends of Stem Cell Extractors

Companies retaining technology of stem cell extraction method anCompanies retaining technology of stem cell extraction method and systemd system

CytoriCytori Therapeutics, Inc.Therapeutics, Inc.MacroporeMacropore BiosurgeryBiosurgery, Inc., Inc.

www.www.cytorixcytorix.com/us.com/us

Tissue Genesis, Inc.Tissue Genesis, Inc.

www.tissuegenesis.comOsiris Therapeutics, Inc.Osiris Therapeutics, Inc.

www.www.osiristxosiristx.com.com

Retaining Retaining TechnologyTechnology

(Patent)(Patent)

System and method of isolation and concentration of regenerative cells from

tissues and others. PCT No. : WO 2005/012480

Applied : June 25, 2004

Method and apparatus of preparation for tissue transplantationPCT No.: WO 200709036

Applied : July 12, 2006

Isolating, purifying and culturing method of human mesenchymal stem

cell( hMSC) and others.US Patent No.: US5827740

Applied : July 30, 1996

Condrogen, Osteocel

With various methods, trial of isolation of stem cells extracted from

bone marrow. (antigen-antibody response)

Isolation of a certain stem cells using antigen-antibody response

ProductProduct CelutionTM TGI 1000 system, TGI 1001 system

PrinciplePrinciple

Consisting of collecting chamber and processing chamber. Processing chamber

can apply filters to remove impurities from fat cell mixtures during

centrifugation and collect stem cells.

Applying filtering and centrifugation simultaneously, can isolate stem cells.

Similar to Cytori’s CelutionTM

FunctionFunction During Centrifugation, extraction of stem cells using multi-staged filters.

As an isolation method of stem cells using filter and centrifuge, can also

perform even implantation at a time

In domestic market, development technology for stem cell isolation systems is at lower level.

http://www.cytoritx.com/us/products/cs_overview.html

01. 01. Bio MasterBio Master의의 개요개요Current Technological Trends of Bioreactor

The current technologies of Bioreactors have been world-widely developed.

Commercialization of Bioreactor was made by Aastrom, an USA-based company in mid 1990.

In recent time, Bioreactors with nano scale for stem culture are under research.

Cellforcel, an automated bioreactor, was developed by Cell-force, a Japanese company (www.cell-force.com).

In Korea, reportedly Biotron Company has developed an bioreactor for stem cell culture (www.Biotron.com).

http://www.cell-force.com/


Major Technologies of Medi-khan

01. 01. Bio MasterBio Master의의 개요개요Major Technology of Medi-khan

Lipokit

Cell TherapysystemCell TherapysystemAutomated Extractor

Of Stem CellsAutomated Extractor

Of Stem CellsHarvesting Machine

of pure fat tissuesHarvesting Machine

of pure fat tissues

• Trial of massive culturing of stem cells through 3 dimensional cell culturing technology. • Introduction of stem cell culturing technology to stem cell extractor is under process. • With fully automated system of stem cell isolation and culture, our advanced technology can be applied for stem cell production for cell therapy.

Fat Transfer Device integrating liposuction and centrifugation in one system Fast & Massive Fat Transfer can be performed.Can harvest pure, concentrated fat tissues using

closed-circuit AFT syringes equipped with weight-mesh piston to remove free oils and prevent contamination.

KFDA Approved (2004) w/ proprietary patentSales Contract with Biomaster in JapanDomestic Sale to Plastic & Cosmetic SurgeonsDomestic(15) and overseas(11) patent protected

• In 2006, development of automated stem cell extractor.• Can automatically isolate stem cells from fat tissues at fast speed. •Can separate stem cell with high yield rate at 37℃ .•All processes are automatically made.• 1st Quality test was finished w/ enhancing its performance. •Pending Patent (#20-2004-0010685) –Centrifuging unit integrating liposuction and fat injection. And other patent (Domestic 9, Overseas 9)

Automated Stem Cell Extractor (YC-100) Cell Therapy System

2001 2004 2007 2010

01. 01. Bio MasterBio Master의의 개요개요Patents for Automated Stem Cell Extractor(YC-100)

Centrifuging Device

Centrifuging Device & MethodCentrifuging Device & Method

Application No. : 10-2006-0111264, Applied Date : Nov. 10, 2006Summary: Device & Method featured by angle control part to control a receptor angle so that collected top layer fluids during centrifugation can flow down at over 90 angle.

Application No. : 10-2006-0060513, Applied Date: June 30, 2006Summary : Centrifugal device having a conical shape to move up materials on the receptor to the top layerby centrifugal force and also having a filter on the top layer of the receptor to pass only some parts of thematerials through.



Application No. : 10-2006-0111266, Applied Date : Nov. 10, 2006Summary: Centrifugal device and method to distinguish and separate fluids by differences of large or small Centrifugal forces and resulting control of revolution speed. Based on the principle that magnitude of centrifugal force by revolution speed differs between the specific components of fluid materials.

Application No. : 10-2006-0111265, Applied Date : Nov. 10, 2006Summary: Centrifugal device & Method to exactly and easily distinguish and recover fluid layers by difference of specific gravities and fluid resistance between the separated fluid materialswhen opening part of the container is opened after revolution body is revolved by centrifugation.

01. 01. Bio MasterBio Master의의 개요개요Technological Value of Stem cell Extractor, YC-100(International Search Report)

CYTORI THERAPEUTICS TISSUE GENESIS

MEDIKAN YC-100


Automated Stem Cell Extraction & Culture Device (YC-100)

01. 01. Bio MasterBio Master의의 개요개요Automated Stem Cell Extraction & Culture Device (YC-100)

Basic SpecificationBasic SpecificationBasic Specification External Design of YC-100 External Design of YCExternal Design of YC--100 100

구 분

Items Specifications

Max RPM 3,000 rpm

Max Capacity 2,000 ml

Max centrifugal force >200g

Rotor radius of rotation range 0.1 rpm

Drive system Ultra-precision Servo Drive control

Speed preset and display 1 rpm

Temperature Range -20 ℃ ~ +50 ℃

Timer 99 hrs 59 min 59 secAnd “Hold” for free run

Acceleration Time 3 stage

Deceleration Time 10 setting

Programmability 100 memory

Digital Display RPM, RCF, Time, Temperature, Temp, Limit, Program, Acel/Decel

Power Requirements 220 V, 60 Hz, single phase, 3.0 kw

Dimension 1310 W×620 D×1230 H / mm

As the 1st stage for Automated Cell Culture System, achieved at automation of whole process of stem cell extraction. With no separate work or additional process, can automatically separate stem cells at high speed with just one consecutive process for

experimental or therapy purpose.



Infusion Apparatus of Fat Tissues & Reagents• Control of infusion & discharging quantities using roller pump and pneumatic apparatus

Using simultaneously operative Upper and Lower Infusion Tubes, can infuse or discharge required fat tissues or reagents even during operation.

Can control temperature at the mount for Fat tissue and Reagent Container

Can sense the weight of every infusion material or reagent using load-cell.

Use of disposable silicon tube



Touch-Pad Monitor• Window Garbin (가빈 ?) User - Graphic User Interface + Touch Screen

Key Pad : Supplementary input apparatus

With employment of touch-pad monitor, can easily and simply set up required programs and operate YC-100.


Built-in Revolutionary Chamber• Chamber with non-uniform plane against centrifugal forces

• Can isolate large quantity of stem cells at a time unlike the existing extractors.

• Using difference of centrifugal force and gravity at each point, can collect stem cells at end of cone.

• With employment of double-step tilting function to the cones, can isolate stem cells very well

• Using disposable membrane within chamber, can prevent any possible contamination during isolation.



Air Handling Unit (Heating)

Can control even minute temperatures using air conditioner and hot air blower

Can remove contaminated materials with circulatory filtering of inner air flow.


Strobo & Moving Image Camera

Synchronization of intermittent on/off timing on revolution of revolving chamber

Can observe the end part of the cones of the chamber with naked eye even during high-speed revolution.

Can shoot both stopped and moving images of rotor (?) using high-tech moving image camera

Can observe the whole process of stem cell isolation

Automated Stem Cell Extraction & Culture System, (YC-100)

Stroboscope Light

Moving Image Camera

01. 01. Bio MasterBio Master의의 개요개요Automated Stem Cell Extraction & Culture System, (YC-100)

Other Functions of YC-100

Based on embedded CPU, employing high-end PC

Graphic-based Interface for user-friendliness

Earth Leakage Circuit Breaking Function

Proprietary Patented Automation System

01. 01. Bio MasterBio Master의의 개요개요Efficiency Test for Stem Cell extraction & Culture System, YC-100

Harvesting fat tissues by Lipokit (made by Medikhan Corp.)

To minimize errors, efficiency test with only highly concentrated fat tissues having over 90% purity to be harvested by Lipokit

vs.

Manual Method: manual isolation of stem cells from fat tissues directly by skilled expert

Automated Method: isolation of stem cell by YC-100

Collagenase treatment

Separation of fat tissues by centrifuge

Washing by PBS buffer

Cell harvest

Cell counting

Comparison & analysis of efficiency rates of stem cells isolated by experts and by YC-100

Analysis of over 3 times per session using same fat tissues

Testing totally over 20 sessions of efficiency rates

Counting the number of total cells (ADSCs & fibrobast etc.) isolated from fat tissues by automatic cell counter after staining with PI (Propidium Iodide)


　 Manual Automated

Cell number(0.1x million cells/fat1cc)

9.65 10.9

Yield (%) 100 112.84

0

20

40

60

80

100

120

140

수동 자동

Isolation method

Isol

atio

n yi

eld

(%)

Efficiency Test of Stem Cell Isolation by YC-100

manual Automated

Efficiency Rate for Stem Cell Isolation by YC-100 will be continuously tested and enhanced.

Characterization (like FACS analysis, proliferation and differentiation) of isolated adipose-derived stem cells will also be identified.

01. 01. Bio MasterBio Master의의 개요개요Processing for Isolation of Adipose-derived stem cell (a few processes are intentionally omitted. Confidential.)


Potential Prospect of YC-100

01. 01. Bio MasterBio Master의의 개요개요Development of Culture Technology

Development of scaffolds with various sizes and shapes Development of Scaffolds for Injection Based on PLLA scaffolds, mixed scaffolds with various biological synthetic

materials are under development.Together with stem cell extraction & culture system, efforts for high

efficiency of stem cell culture and proliferation will be continued.

x200

0

5

10

15

20

25

30

35

40

45

50

initial 6h 3d 6d 10d

Culture time

Cell

num

ber

(1X

105

cells

/bea

ds 1

cc) PLLA beads

Fat scaffold

Dextran beads

Retaining Patents (Domestic 13, Overseas 6)

10-2002-0001131 : As an injection material, solid chitosan powder with sponge structure to be injected for human body and its production method and injection apparatus.

10-2002-0025375 : As an injection material, solid chitosan powder with sponge structure to be injected for human body and its production method

10-2002-0064189 : In Vitro Culture Method of human cells.

10-2002-0085331 : 3 dimensional cell culture method using solid powder

10-2005-0078382 : As an injection material, solid chitosan powder with sponge structure to be injected for human body and its production method

10-2006-0079725 : Micro Scaffolds with non-spherical 3 dimensional stereo structure to be produced by rapid prototyper and used for cell culture and transfer.

10-2007-0041058 : Cell culturing scaffold with increased specific gravity and its production method.

< In vitro proliferation test using ADSC P0 >

Development of 3 Dimensional scaffolds for cell proliferation

01. 01. Bio MasterBio Master의의 개요개요Stem Cell Culturing System

<Simulation for 3 Dimensional Stem Cell Culture >


Dreaming of Future High-tech Bio Business

MedikhanDevelopment of automated Stem Cell Extraction & Culture Device

Exploitation of Cell TherapyCell Banking Business

Biomaster (Japan)

Clinical Research using Stem Cells

(Extraction of Stem Cell + Bioreactor + Cell therapy)

Potential Prospect of YC-100 Stem Cell Extraction and Culture Device

01. 01. Bio MasterBio Master의의 개요개요FDA regulation on HCT/P’s

01. 01. Bio MasterBio Master의의 개요개요FDA regulation on HCT/P’s


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Automated Extraction & Culture System of Adipose …doc.xueqiu.com/144d468295a2f23f9789fc75.pdf · Automated Extraction & Culture System of Adipose-derived Stem Cells ... Potential