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Komang Ardi Wahyuningsih

Prof. dr. Jeanne Adiwinata Pawitan, PhD


DR. Puji Lestari, M.S
dr. Reza Yuridian Purwoko,
Sp.KK
Evah Luviah

Introduction

Regenerative medicine multidisciplinary

science:

- biomaterials,
- growth factors, &
- stem cells to repair failing organs
ADSCs stem cell source w/ therapeutic
applicability in diverse fields repair &
regeneration of acute & chronically damaged
tissues

ADSc = BMSC
cell surface marker
gene expression profile
differentiation potential
ADSc >> BMSC
obtained in large quantities @ low risk
yields far more stem cell than BM (5000:100-

1000)

Obtained from liposuction aspirates &

differentiate into multiple lineages of


mesodermal or ectodermal origins.
It shown differentiation into
- adipogenic,
- osteogenic,
- chondrogenic,
- myogenic,
- cardiomyogenic, &
- neuro-genic lineages

Valuable source of ADSc WAT


ASCs can also be harvested by a minimally

invasive procedure involving liposuction from


subcutaneous depots.
Main depots of WAT :
subcutaneous depots in the buttocks, thighs &
abdomen
visceral/intrabdominal depots around the
omentum, intestines and perirenal areas.

The stem cell population derived from

collagenase-digested human adipose tissue


(SVF)
differentiate into multiple cell lineages,
including the adipose tissue, cartilage, bone,
skeletal muscle,neuronal cells, endothelial
cells,cardiomyocytes, & smooth muscle cells

Isolation Method for ASCs


We obtained ADSc from liposuction aspirates

collected using both manual canula & PAL


(Microaire).
Lipoaspirate was obtained by tumescent
liposuction and kept in a sterile bottle at 4C
for no more than 24 hours.

A. Manual canula, there are 4 type of canula with total 30


holes, 5 holes for each side, with 1 mm hole diameter, &
length of range 200-230 mm.
B. PAL-canula there are 10 type of canula with total 6 holes,
1-2 holes for each side, with 8-10x3-5 mm hole length x width,
& length of range 220-300 mm

All protocol for preparation of ADScs isolation

based on method from Pawitan et al., 2012.

Lipoaspirate obtained from PAL and manual (1) were filtered through a fine mesh stainless steel tea or coffee filter (=7 cm) to separate adipose tissue
fragments from tumescent, blood, or contaminants (2), and then wash the fat tissue fragments by soaking the filter in a sterile porcelain bowl filled with
Phosphate Buffer Saline (PBS 1x) pH 7.4 and stirring using a small tea spoon to remove the other contaminants (3). This washing step is repeated
several times. When fat tissue fragments appeared yellow and clean, then transferred into sterile 50 ml centrifuge tube (4). 0.075% collagenase type 1
(Sigma, USA) is added into the tube containing fat tissue fragments until tissue fragments : collagenase solution was 1:2 (f5). The tubes then
incubated at 37oC, 5% CO2 for 1 hour with agitation every 5 minutes. After 1 hour, the appeared floating yellow free lipid were removed (6 & 7) and
infranatant were poured into new centrifuge tube through nylon mesh filter and divided into sterile 15 ml centrifuge tube (8 & 9) and then centrifuge
1200 rpm for 10 minutes. After centrifugation step, the sedimented pellet were added lysis buffer if red blood cell still appeared (10 &11), then
centrifuge step repeated, obtained pellet (12) then resuspended in culture medium. The number of viable cell were counted using Hemacytometer.
The cells were cultured in 12-well plate with seeding number around 170.000 viable cells per well). The cultures cells were observed and medium
changed every 2 3 days.

Description

CANUL
A

MICROA
IRE

Lipoaspiraate

Dark
red

Bright
red

Isolation
processing
time/washing
process in the lab

>>

>>>

Sample after
washing process
with PBS several
times

Bright,
Yellowis
yelowwi h, still
sh
seems
reddish

Remark

Description

CANUL
A

MICROAIRE

Sample after
digestion with
Collagenase

No RBC

Still there is
RBC (RBC
still present
while filtering
infranatant
through
nylon mesh
filter)

Cell pellet and


after seeding >
2 days

Clearer

the fat still


present in
pellet

Remark

Day-2
Manual
Canula

Microair
e

Day-4

Day-6

Day-9

Patient ID

Age

Sex

BMI

Harvest site

24

55

23.76

25

39

29.1

26

55

25.45

27

32

29.1

Abdomen

28

41

41.24

Abdomen

29

42

28.85

Abdomen

Flank
Abdomen
Flank

Patient ID

Age

24

Sex

BMI

23.76

Harvest site

Flank

Patient
ID
24

55
25

29.1

Abdomen

25

25.45

Flank

26

27

29.1

Abdomen

27

28

41.24

Abdomen

28

41
29

42

28.85

Abdomen

29

300 ml

1,660,000

11 G

300 ml

2,460,000

4.0 mm

300 ml

900,000

11 G

300 ml

1,310,000

4.0 mm

100 ml

855,000

11 G

300 ml

1,920,000
1,883,333

4.0 mm

500 ml

11 G

<500 ml

5.00 mm

>300 ml

11 G

>300 ml

4.0 mm

300 ml

11 G

250 ml

PAL
Manual Canula

3.0 mm

PAL
Manual Canula

32

Cell yield
count

PAL
Manual Canula

55

Lipoaspirate
Volume

PAL
Manual Canula

39

Canula type

PAL
Manual Canula

26

Method

2,885,000

PAL
Manual Canula

1,593,333

2,380,000
890,000
910,000

Age

: no differentiation between all ages

varian
Sex
: Female greater in number of cell
yield count than male
Harvesting site : ADSc from flank greater in
number of cell yield count than from abdomen
Canula type : cell yield from manual canula
lipoaspirate greater in number than PAL
lipoaspirate.

soft and stable small fat particle

including many stromal cells


Highest density fat fraction
contain more progenitor cells
and increased concentrations of
several angiogenic/vasculogenic
and anti-inflammatory cytokines

Correction of dark circle on lower eyelid


by structural high density fat graft,
Sung Min Kim, M.D.

Immunoprivileged << expression of MHC-II

& co-stimulatory molecules on the cell surface


allogeneic transplantation into
immunocompetent recipients
Immunomodulator & can promote tissue
repair prevent & treat acute GVHD in
allogeneic stem cell transplantation,
autoimmune diseases & inflammatory
diseases.

Characterization of ADSc

Secrete an array of soluble factors promote

tissue regeneration
The secretome includes angiogenic factors
(VEGF), anti-apoptotic factors (IGF-1),
hematopoietic factors (colony stimulating
factors & interleukins), & HGF

Differentiate into several lineages of

osteogenic, chondrogenic, adipogenic,


cardiomyogenic, myogenic,& neurogenic cells.
Also into tissues of endo- & ectodermal
lineages such as hepatocytes, pancreatic islet
cells, endothelial cells, neural cells, &
epithelial cells too

ADSc application on
esthetics
Soft tissue augmentation
The augmentation of breast was successful &

had satisfactory clinical results w/out any


major complications
depressed scars
Wound healing
diabetes wound
Hair follicles regeneration

Conclusion
in regenerative medicine

Adipose-derived stem cells play an important role of


Because adipose-derived stem cells demonstrate

several interesting properties, suggests potential


clinical promise in many medical disciplines.
Protocol for adipose-derived stem cell use or clinical
application in terms of type of cells used (stromal
vascular fraction cells or cultured & purified adiposederived stem cells) become easily available & no
money & no time consuming.
Further basic science experimental studies still
needed to be performed to ensure the safety &
efficacy of ADSc

Reference

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Thankyou