ORIGINAL RESEARCH ARTICLE Open Access
Cy okine-Rich Adipose Tissue Ex ac P oduc ion
om Wa e -Assis ed Lipoaspi a e:
Me hodology o Clinical Use
Jenny Lopez,
1,2,
*Ou i Hu ala,
3
Je a-Riina Sa kanen,
1,4
Ilkka Kaa inen,
2,4
Hannu Kuokkanen,
2,4
and Timo Ylikomi
1,4
Abs ac
P ope unc ioning wound healing s a egies a e spa se. Adequa e ascula o ma ion o he inju ed a ea, as
well as eplacemen o he olume loss, is undamen al in so issue epai . Tissue enginee ing s a egies ha e
been p oposed o he ea men o hese inju y si es. No el cell- ee subs ance, human adipose issue ex ac
(ATE), has been p e iously shown o induce in i o angiogenesis and adipogenesis and in i o so issue o -
ma ion. This s udy epo s he ansla ion o ATE p epa a ion om labo a o y o he ope a ing oom (OR).
ATE samples o his s udy we e de i ed om adipose issue ob ained wi h he wa e -je assis ed liposuc ion
echnique om 27 heal hy pa ien s. The a iables s udied included incuba ion ime (15, 30, and 45 min), em-
pe a u e ( oom empe a u e s. 37C), and fil e ype o de e mine he op imal me hod yielding he mos con-
sis en o al p o ein con en , as well as consis en and high exp ession o adipose-de i ed g ow h ac o s and
cy okines, including: ascula endo helial g ow h ac o , basic fib oblas g ow h ac o , in e leukin-6, adiponec-
in, lep in, and insulin-like g ow h ac o . Following he op imiza ion, samples we e p oduced in he OR and
es ed o hei s e ili y. No significan di e ences we e obse ed when compa ing ex ac incuba ion ime
poin s o incuba ion empe a u e. None heless, when s udying he di e en fil e ypes used, a sy inge fil e
wi h PES memb ane wi h la ge fil e a ea showed significan ly highe p o ein concen a ion ( p£0.018).
When s udying he di e en g ow h ac o concen a ions, ELISA esul s showed less a ia ion in cy okine con-
cen a ions in he OR samples wi h he op imized p o ocol. All o he OR samples we e es ed s e ile. The de-
ised p o ocol is an easy and ep oducible OR- eady me hod o ATE gene a ion. As an a ac i e sou ce o
g ow h ac o s, ATE is a p omising al e na i e in he as field o issue enginee ing. I s clinical applica ions
include olume eplacemen as a complemen o fille s and imp o emen o he pe manence o a g a s
and wound healing, among o he bioac i e unc ions.
Keywo ds: acellula biological ma ices; adipose; angiogenesis and asculogenesis; biomedical enginee ing;
g ow h ac o s
In oduc ion
Cu en ly, he majo challenge in issue enginee ing lies
no only in he high cos s o ma e ial p oduc ion and
sa e y o he bioma e ial used bu also mos impo -
an ly in he lack o e ficacy in p omo ing ascula
o ma ion and so - issue eplacemen .
1–4
In pa icula ,
neo ascula iza ion induc ion is a majo obs acle o de-
eloping issue enginee ing s a egies.
5–9
Ma u e human adipose issue, conside ed an endo-
c ine en i y on i s own, is a known sou ce o g ow h
1
Depa men o Cell Biology, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland.
2
Depa men o Plas ic Su ge y, Uni o Musculoskele al Diseases, Tampe e Uni e si y Hospi al, Pi kanmaa Hospi al Dis ic , Tampe e, Finland.
3
FICAM, Finnish Cen e o Al e na i e Me hods, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland.
4
Science Cen e , Pi kanmaa Hospi al Dis ic , Finland.
*Add ess co espondence o: Jenny Lopez, MD, Depa men o Cell Biology, School o Medicine, Uni e si y o Tampe e, PL100, 33014 Tampe e, Finland, E-mail:
[email p o ec ed]
ªJenny Lopez e al. 2016; Published by Ma y Ann Liebe , Inc. This Open Access a icle is dis ibu ed unde he e ms o he C ea i e Commons License
(h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal
wo k is p ope ly c edi ed.
BioResea ch Open Access
Volume 5.1, 2016
DOI: 10.1089/bio es.2016.0030
BioResea ch
OPEN ACCESS
269
and di e en ia ion p omo ing ac o s.
10–16
These
g ow h ac o s ha e he abili y o induce p oli e a ion,
mig a ion, and di e en ia ion o a ious cell lines.
13–17
Ex ensi e unc ions and clinical applica ions o g ow h
ac o s a e well epo ed in he li e a u e.
18–24
Adipose
issue is an excellen sou ce o adipose s em cells in ad-
di ion o he adipose-de i ed g ow h ac o s,
11,25–30
and human adipose issue is easily a ailable h ough
liposuc ion.
We ha e p e iously de eloped an acellula bioac-
i e ex ac om ma u e adipose issue, p o en o
con ain impo an adipose issue cy okines, including
ascula endo helial g ow h ac o (VEGF), basic fi-
b oblas g ow h ac o be a (FGFb), in e leukin 6
(IL-6), insulin-like g ow h ac o 1(IGF-1),adipo-
nec in, and angiogenin, among o he s.
31,32
This ex-
ac demons a es a unique capaci y o inducing
angiogenesis and adipogenesis.
31
Animal expe imen al models indica ed ha , in com-
bina ion wi h hyd ogel, his adipose issue ex ac
(ATE) p omo ed neo ascula iza ion and so issue ex-
pansion. In addi ion, i was shown ha he pe manence
o he e ec o he ex ac emained o 9 mon hs. Fu -
he mo e, du ing sho - and long- e m ollow-up, no
hype sensi i i y o o eign body eac ions we e epo ed
wi h human ex ac in a expe imen al models.
32
When aiming a using his au ologous ex ac in
clinical s udies, he de elopmen o a s aigh o wa d
and eliable me hod o ope a ion oom p epa a ion
is essen ial. ATE was p e iously p oduced om solid
ma u e adipose issue and shown o be bioac i e
in i o and in i o.
31,32
In he cu en s udy, ATE
was p oduced om lipoaspi a e ma e ial. This simple
su gical p ocedu e and ope a ing oom (OR) p epa a-
ion would make ATE an a ac i e sou ce o mul iple
g ow h ac o s and cy okines wi h a ple ho a o u-
u e applica ions.
32
In i o, hese g ow h ac o s ha e demons a ed he
induc ion and di e en ia ion o adipocy es, endo he-
lial cells, ke a inocy es, chond ocy es, and os eoblas s,
among o he s in ol ed in issue egene a ion.
31,33,34
The po en ial o ATE o di e en applica ions holds
alue in a eas whe e so issue o ma ion and olume
a e equi ed, especially in olume loss and po en ially
o op imizing a g a ing pe manence.
32
In his s udy, he op imal me hod o human a ex-
ac p epa a ion was in es iga ed wi h di e en a i-
ables, such as incuba ion ime, empe a u e, and fil e
ype. These cha ac e is ics we e conside ed key when
ans e ing he me hodology om he labo a o y o
he OR while mee ing he clinical and su gical s an-
da ds. The goal was o de elop a p o ocol ha mee s
clinical s anda ds ha allow he su geon o p epa e
he ATE in he OR o immedia e applica ion and
ansla e he ATE labo a o y p o ocol o clinical he -
apeu ic use.
Ma e ials and Me hods
E hical aspec s
The s udy was conduc ed acco ding o he Decla a ion
o Helsinki, he Eu opean Guidelines on Good Clinical
P ac ice, and was app o ed by he E hics Commi ee o
he Pi kanmaa Hospi al Dis ic , Tampe e, Finland
(R03058). The human adipose issue samples we e
ob ained om su gical ope a ions wi h in o med con-
sen s a he Tampe e Uni e si y Hospi al, Tampe e,
Finland.
Samples
Human adipose issue samples we e ob ained h ough
he wa e -assis ed liposuc ion echnique (body-je ;
Human Med AG, Ge many) and p ocessed unde s e -
ile condi ions. A o al o 27 pa ien s we e included in
he s udy. The main indica ions o liposuc ion in
hese pa ien s we e a g a ing and body con ou ing.
Exclusion c i e ia o he dono s we e pa ien s ecei -
ing ho monal he apy, a e cance abla i e su ge y,
ac i e cance , ecen chemo he apy ea men , and
como bidi ies ha con aindica ed he su ge y. All su -
ge ies we e une en ul, and no complica ions we e
epo ed. All es ed samples we e om emale pa ien s,
and hei mean age, weigh , and body mass index a e
lis ed in Table 1. Lipoaspi a ion was pe o med in he
abdominal subcu aneous issue in 76.2% o he pa-
ien s, om he flank egion in 19% and om he
high in 4.7%.
Lipoaspi a ion p ocedu e
Liposuc ions we e pe o med unde gene al o spinal
anes hesia using he wa e -assis ed echnique (body-
je , Human Med AG, Ge many). Tumescen solu ion
con aining 1 mg o ad enaline and 250 mg o lidocaine
pe 1000 mL saline was infil a ed. The p essu e o in-
fil a ion and suc ion was done a a ange o 2 o 3 (50
Table 1. Pa ien Demog aphics
Mean alue Range
Age 51.95 33–68
Weigh 77.1 57–94
Body mass index 28.1 21–38
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270
o 70 ba ). Lipoaspi a ion was pe o med unde
400 mba p essu e. As a guide, ange 2 is equi alen
o 110 mL/min and a ange 3 o 130 mL/min o umes-
cen je emission. Adipose issue collec ion was done
unde a s e ile en i onmen in o a canis e (LipoCol-
lec o , Human Med, Ge many) and ans e ed o
50 mL sy inges. The amoun o a aspi a ed o sam-
pling a ied om 40 o 100 mL pe pa ien .
P oduc ion o ATE in he labo a o y
In he labo a o y, ATE was p oduced by adding Ringe
lac a e (Bax e Heal hca e Co po a ion, Helsinki, Finland)
o he adipose issue sample a an app oxima e a io o 1:1
and hen p ocessed acco ding o he di e en s udy a i-
ables. Incuba ion was pe o med a 37C wa e ba h o
a oom empe a u e (RT). The incuba ion imes s udied
we e 15, 30, o 45 min. A e incuba ion, he samples
we e s e ile fil e ed wi h di e en 0.2 lm po e size sy inge
fil e s (Ac odisc

fil e , polye he sul one PES memb ane
[PALL Li e Sciences, New Yo k]; Minisa NML fil e ,
cellulose ace a e memb ane [Sa o ius AG, Ge many];
Fil opu S Plus fil e , cellulose ace a e memb ane [Sa -
s ed & Co, Ge many]; and Millex GP fil e , PES mem-
b ane [Me ck, Millipo e, Ge many]). Once fil e ed, he
ATE was s o ed a 20C un il sample analysis.
P oduc ion o ATE in he OR
ATE p oduc ion was pe o med on a sepa a e s e ile
bench in he OR. A e pe o ming he lipoaspi a ion,
he adipose issue was gen ly mixed wi h p ewa med
(37C) Ringe lac a e solu ion a an app oxima e a io
o 1:1. The mix u e was incuba ed o 30 min a RT.
The lowe laye con aining he Ringe lac a e solu ion
was passed h ough a s e ile fil e and ozen a 20C
un il u he use. The p epa a ion me hod o ATE is
summa ized in Figu e 1. ATE samples ha e p e iously
been shown o induce adipogenesis om 200lg/mL up-
wa ds in cell cul u e.
31,32
Thus, o al p o ein concen a-
ion o 200 lg/mL was selec ed as he lowes accep ance
limi o ATE samples. ATE samples p epa ed in he lab-
o a o y and OR o igina ed om di e en dono s.
S e ili y es o OR samples
S e ili y es was pe o med om six ATE samples p e-
pa edin heOR.App oxima ely,2mLo ATEwas
added pe BacT/ALERT sys em (bioMe
´ ieux SA, F ance)
bo le. Ae obic BacT/ALERT SA and anae obic bac e ia
es ing BacT/ALERT SN (i.e., g am-posi i e and g am-
nega i e bac e ia and yeas ) we e pe o med o each
ATE sample. Samples we e cul u ed in BacT/ALERT
3D (bioMe
´ ieux SA) o 10 days be o e analyzing bac e-
ial g ow h. ‘‘Nega i e’’ esul s in he sys em indica ed
ha he e was no bac e ial o yeas g ow h.
Measu emen o p o ein concen a ion
To al p o ein con en o he samples was measu ed using
Pie ceBCA P o ein Assay Ki (The mo Scien ific, Wal-
ham, MA) acco ding o manu ac u e ’s ins uc ions
using bo ine se um albumin (BSA) as a s anda d. Resul s
we e measu ed a e 30 min incuba iona 37Ca 562nm
wi h Va ioskanFlash Mul imode Reade (The mo Sci-
en ific).
Measu emen o g ow h ac o concen a ion
Ex ac samples we e es ed wi h colo ime ic sandwich
ELISA, Cus om made ELISA s ips (Signosis

,San a
Cla a, CA). The ollowing cy okines we e e alua ed:
VEGF, umo nec osis ac o alpha (TNFa), in e e on
FIG. 1. S eps o ATE p epa a ion. S ep 1, Liposuc ion and collec ion o adipose issue wi h wa e -assis ed
liposuc ion. S ep 2, T ans e o a in o sy inges and addi ion o Ringe solu ion. S ep 3, 30 min incuba ion and
fil a ion o p oduce s e ile ATE, which is eady o clinical use. ATE, adipose issue ex ac .
Lopez, e al.; BioResea ch Open Access 2016, 5.1
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271
gamma (IFNc), g anulocy e colony s imula ing ac o (G-
CSF), g anulocy e mac ophage colony s imula ing ac o
(GM-CSF),IL-6,IL-8,IGF-1,IL-1a,FGFb, esis in,mac-
ophage inflamma o y p o ein (MIP-1), adiponec in, lep-
in, and an es. P o ein s anda ds o cus om human
cy okine ELISA S ip (Signosis) we e used wi h concen-
a ions o 2 and 1 ng/mL. The ELISA s ips we e used
acco ding o manu ac u e ’s ins uc ions as ollows. To
each well, 100 lL o he s udied ATE ba ch was added
and incuba ed o 1 h wi h gen le shaking in RT. The liq-
uid was hen aspi a ed om each well and he wells we e
washed h ice wi h 200 lL o assay wash bu e pe well.
Subsequen ly, 100 lLo S ep a idin-HRPconjuga edi-
lu ed 1:200 in diluen bu e was added o each well and
incuba ed o 45 min a RT unde gen le shaking. A e
incuba ion, he wells we e washed h ice wi h 200 lLo
washing bu e . One hund ed mic oli e s o subs a e
was added in each well and hen incuba ed be ween 5
and 30 min pe cy okine. The eac ion was ended
wi h he addi ion o 50 lLo s opsolu ion oeach
well ow simul aneously and de ec ing a isible colo
change o he s anda d. The op ical densi y was de e -
mined a 450 nm wi h Va ioskan Flash mul imode
eade (The mo Scien ific).
S a is ical analyses
S a is ical analyses we e pe o med and g aphs p o-
cessed wi h G aphPad P ism 5.0 (G aphPad So wa e,
Inc., San Diego, CA). The esul s we e epo ed as
mean –SD, and di e ences we e conside ed significan
when p<0.05*, p<0.01**, and p<0.001***. Resul s o
o al p o ein concen a ion in incuba ion empe a u e
compa ison and labo a o y e sus OR p oduc ion o
ATE we e analyzed wi h s uden ’s - es and wo- ailed
pos es . The analyses o incuba ion ime and fil e ype
used we e pe o med using One-way ANOVA wi h
Tukey’s pos es . The ela ionship o g ow h ac o con-
cen a ions in OR samples o labo a o y samples was
calcula ed wi h Pea son’s co ela ion and esul s
depic ed as alues.
Resul s
The e ec o incuba ion empe a u e
The e ec o incuba ion empe a u e on ATE p o ein con-
cen a ion was s udied wi h a cellulose ace a e memb ane
fil e (Sa s ed , Ge many) used in ou p e ious in i o
s udies.
31
No significan di e ence in o al p o ein con-
cen a ion was obse ed be ween RT incuba ion and
37C wa e ba h incuba ion (Fig. 2A). Howe e , he e
was a sligh ly highe concen a ion o o al p o ein in
wa e ba h incuba ed samples compa ed o RT incuba ed
samples.
The e ec o incuba ion ime
Incuba ion imes o 15, 30, and 45 min we e s udied o de-
e mine he sho es ime o OR ATE ex ac ion (consid-
e ing possible immedia e clinical use) in e e ence o
p o einyield.Incuba ionwaspe o medinRTand heex-
ac passed h ough a cellulose ace a e memb ane fil e
(Sa s ed , Ge many).
31
The o al p o ein concen a ion
be ween he ime poin s did no di e significan ly (mul-
iplici y adjus ed p‡0.992, Fig. 2B), bu he p o ein yield
showed less a ia ion and sligh ly highe alues in he
30 min ime poin .
Fil e es ing
To es ima e whe he he p o ein yield a ied among
di e en fil e memb anes, ou di e en 0.2 lm fil e s
we e selec ed. ATE was p oduced a 30 min incuba ion
a RT and subsequen ly s ained h ough he fil e s.
Close o 1000 lg/mL p o ein was ob ained wi h all fil-
e s, bu wi h polye hylene sul one (PES) memb ane
and la ges su ace a ea, showed a significan ly highe
p o ein yield (mul iplici y adjus ed p£0.018) com-
pa ed o he o he fil e s (Fig. 3).
T ans e o he ex ac ion p o ocol
om labo a o y o he OR
To ans e he labo a o y me hodology o he OR,
ATE was p oduced a RT incuba ion o 30 min and
subsequen ly fil e ed wi h PES memb ane. The esul s
in Figu e 4 show ha he samples p oduced in he lab-
o a o y had g ea e de ia ion ye highe p o ein con-
cen a ions. Howe e , hese we e no significan ly
highe han he OR samples ( p0.0730, medians 1210
and 321.5, espec i ely).
G ow h ac o con en o he samples
To con ol he quali y and assu e he bioac i i y o he
samples, g ow h ac o measu emen s o bo h labo a o y
and OR samples we e s udied. The esul s show ha he
g ow h ac o yields we e compa able be ween he sam-
ples p oduced in he labo a o y and hose om he OR
(Fig. 5) al hough he co ela ions a ied be ween he
g ow h ac o s (Fig. 5). The samples had less a ia ion
when pe o med in he OR wi h he op imized p o ocol
(Fig. 5). Mos o he g ow h ac o s co ela ed well wi h
labo a o y samples and in addi ion, VEGF (also highe
CV) and G-CSF p oduced highe mean g ow h ac o
concen a ions in he OR wi h he op imized p o ocol.
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272
S e ili y es
A s e ili y es was pe o med o ensu e asepsis o
he samples in he OR. All ATE samples we e
nega i e o bac e ial g ow h in ae obic and an-
ae obic media.
Discussion
One o he main challenges in issue enginee ing is o
disco e a bioac i e sou ce o g ow h ac o s wi h
egene a ing p ope ies while accele a ing local ac ion,
enhancing olume eplacemen and s imula ing he
g ow h o mul iple cell lines.
13,35–38
The mos impo -
an ea u e is o apidly induce issue neo ascula iza-
ion o a oid hypoxia and ischemia.
39–43
Ideally, his
bioac i e ma e ial should also be cos beneficial, easy
o p epa e, and ep oducible. In ou p e ious s udies,
we ha e been able o isola e a no el cell- ee bioac i e
subs ance, ATE, which no only induces angiogenesis
and adipogenesis in i o and in expe imen al models
bu also is ich in g ow h ac o s.
31,32
The ad an age
o he cu en g ow h ac o ma e ial is ha as i does
no con ain cells, i should ha e less immune eac ions
and i s allogeneic use could be possible. This would
ease i s clinical use. As ATE is a la ge mix u e o g ow h
ac o s, i has po en ial o be e bioac i i y. Clinicians
a e s ill s uggling o imp o e adipose issue ans e
iabili y and wo king on ways o imp o e wound heal-
ing, scena ios ha would benefi om g ow h ac o
use.
The main aim o he cu en s udy was o ans e
he me hod o ob aining adipose issue g ow h ac o s
om he labo a o y o he OR o la e success ul clin-
ical use. ATE has p e iously shown o induce so issue
o ma ion when inco po a ed in o hyalu onic acid and
is he e o e a e y p omising ma e ial o so issue e-
placemen and so issue healing.
31,32
The au ho s
compa ed p e iously used pa ame e s, p o ein yield,
and g ow h ac o con en and ocused on modifiable
pa ame e s when ans e ing he me hodology om
he labo a o y o he clinic. The a iables con empla ed
in his s udy we e incuba ion ime, incuba ion empe -
a u e, and fil e ype. Du ing he ansla ion p ocess o
ATE p oduc ion om he labo a o y o he OR, he e
a e high clinical demands o be me no only om
he clinical ma e ials used bu also o make his a sim-
ple me hod om su gical s a ’s poin o iew. Because
in p e ious s udies,
31,32
whole solid a was p ocessed
in he labo a o y o ob ain ATE, i was c ucial o
FIG. 2. (A) P o ein concen a ion ob ained in 37C and RT incuba ion. Compa ison o o al p o ein con en o
he ATE samples in di e en incuba ion empe a u es. ATE was incuba ed o 30 min in 37C o RT and fil e ed
wi h cellulose ace a e Fil e 3. No significan di e ence was obse ed be ween incuba ion empe a u es when
s a is ically e alua ed wi h s uden ’s - es wi h wo- ailed pos es ( p0.7207) and n=3. (B) P o ein
concen a ions ob ained wi h 15, 30, and 45 min incuba ion. Compa ison o o al p o ein con en o he ATE
samples wi h incuba ion imes o 15, 30, and 45 min. ATE was p oduced in equal condi ions excep o
incuba ion ime, ha is, incuba ion was pe o med in RT and fil e ed wi h Fil e 3. No significan di e ence was
obse ed be ween incuba ion imes when s a is ically e alua ed wi h one-way ANOVA wi h Tukey’s pos es
(p0.9923) and n=3. RT, oom empe a u e.
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273

disco e an easy me hod ha would p ese e an ade-
qua e p o ein yield.
This ans e me hod is no only impo an bu also
complica ed and he impac o hese a iables had o
be de e mined. The e o e, ou aim was o main ain
g ow h ac o yields compa able o ou p e ious s ud-
ies, in a epea able scena io. Mo eo e , ou o iginal
s udies we e pe o med wi h solid adipose issue ha
unde wen p ocessing, so we e ified ha he esul ing
ATE would, in ac , yield simila esul s when o igina -
ing om lipoaspi a es. As a lipoaspi a ion me hod, we
used he wa e -assis ed echnique, which has been s an-
da dly used in ou clinic due o i s simplici y, and
demons a ed adipose issue iabili y p ese a ion.
44
Al hough he echnique uses he powe o wa e a ce -
ain p essu es, i is gen le enough o p ese e adipo-
cy es wi h minimal issue inju y o blood loss. In
addi ion, pa ien eco e y is quicke and mo bidi y
lowe han adi ional liposuc ion.
45,46
In he cu en s udy, we op imized he p epa a ion
p o ocol o ATE o ansla e he p oduc ion om he
labo a o y o he OR, s udying h ee main a iables.
To begin wi h, incuba ion ime is impo an conside -
ing he ime he pa ien was in su ge y unde local, spi-
nal, o gene al anes hesia. In ideal condi ions, su gical
ime should be as sho as possible o p e en pa ien
pe iope a i e complica ions and his is he eason
why he ime poin s o 15, 30, and 45 min we e selec ed.
Fu he mo e, because o iginal ATE samples we e incu-
ba ed ei he in wa e ba h o 37C incuba o , we ound
ha compa ing i o RT would o e he su geon an eas-
ie me hod o p oduce ATE. In ela ion o he fil e ,
du ing he ansla ion p ocess, we no iced ha ope a -
ing hea e s use a a ie y o ma e ials o high clinical
s anda d demands. In ou labo a o y s udies, we used
fil e s ha we e no applicable o he OR en i onmen ;
he e o e, he challenge was o es hose ha did mee
hese s anda ds while p ese ing he p o ein yield. The
p o ocol was es ed wi h ou di e en fil e s wi h PES
and cellulose ace a e memb anes. We no iced ha al-
hough a la ge fil e a ea played a ole, i seemed
ha he hyd ophilic low-p o ein binding na u e o
he PES fil e demons a ed ad an ages. Di e en o
FIG. 4. P o ein concen a ions ob ained in OR
e sus labo a o y. Compa ison o o al p o ein
con en be ween he ATE samples p oduced in
labo a o y and hose p oduced in OR. ATE was
incuba ed o 30 min in RT and fil e ed wi h PES
Fil e 1. No significan di e ence was obse ed
be ween he o al p o ein o he wo p oduc ion
condi ions as e alua ed by s uden ’s - es wi h
wo- ailed pos es ( p0.0922) and n‡6. OR,
ope a ing oom.
FIG. 3. P o ein concen a ions ob ained wi h
ou di e en fil e s. Compa ison o o al p o ein
concen a ion when ATE was p oduced wi h
30 min incuba ion a RT using ou dis inc fil e s;
PES Fil e 1 (7.5 cm
2
), cellulose ace a e Fil e 2
(6.2 cm
2
), cellulose ace a e Fil e 3 (5.3 cm
2
), and
PES Fil e 4 (4.5 cm
2
). S a is ical analysis was
pe o med wi h One-way ANOVA wi h Tukey’s
pos es , p<0.05* and p<0.01** and n=4.
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274
p e ious publica ions, we used lipoaspi a e ma e ial
ha con ains pa icles ha easily clog fil e s, which
also led us o choose he adequa e PES fil e ype. How-
e e , we ecognize ha in e pe sonal a ia ions play a
g ea influence in he final p o ein yield. The op imiza-
ion showed o educe he a ia ion be ween he sam-
ples and he specific g ow h ac o concen a ions
we e no los du ing he p o ocol modifica ions. In ad-
di ion, he samples p oduced in he OR passed he s e-
ili y es s pe o med.
By s udying he e ec o incuba ion empe a u e (RT
s. 37C) and ime (15, 30, and 45 min), we showed
ha he p o ocol is flexible enough o be pe o med
in he busy OR se ing wi hou comp omising he qual-
i y o he p oduc .
When s udying he p o ein yield, we obse ed ha
adequa e amoun s we e ob ained in sho incuba ion
imes (15, 30, and 45 min); ye we se led o 30 min
as sligh ly highe p o ein concen a ions we e achie ed
om his ime poin onwa d. In e es ingly, empe a-
u e a ia ions did no seem o a ec o al p o ein
and cy okine yields. We no iced ha al hough he fil e
cha ac e is ics may play a ole, sample handling had an
impo an e ec on he p o ein yield. Samples ha had
o be neglec ed om he s udy (p o ein concen a ion
<200 ng/mL, ou samples) we e pe o med in he lab-
o a o y, and hus, he ime om he su ge y o he
p epa a ion o ATE a ied. This was based on p e ious
s udies whe e bioac i i y was p o en abo e his con-
cen a ion o p o ein.
31,32
None heless, he fil e ma e-
ial o su ace a ea o he fil e may also ha e an impac
on he p o ein concen a ion and, he e o e, on he ea-
sibili y o he ATE p oduc ion p ocedu e.
We obse ed lowe a ia ions in g ow h ac o and
cy okine measu emen s in he samples p oduced in
OR compa ed wi h he labo a o y samples. I is well
known ha in e indi idual di e ences in cy okine con-
cen a ions om eshly p epa ed p ima y cells a e
usually much highe han he a ia ions seen wi h e-
pea ed p epa a ions o he same dono .
47
The e is an
ine i able pa ien - ela ed a ia ion due o he e ec s
o body mass index, age, weigh , como bidi y, and lipo-
suc ion si e ha may a ec p o ein yield, bu he op i-
miza ion o he p o ocol was success ul in dec easing
he sample- o-sample a ia ion. The g ow h ac o e-
sul s a e also in conco dance wi h ou p e ious s udies
a 1-h incuba ion.
32
Since ATE is p epa ed om au ologous adipose is-
sue, heo e ically he e a e no isks o disease ans-
mission, immunogenic eac ions, o cance . This
solu ion is elu ed om he fil e , p o iding a cell- ee
en iched ac ion o biologically ac i e media o s
(VEGF, IGF-1, e c.), mos o which a e key in wound
healing. The mos abundan adipokines eleased by ad-
ipocy es, lep in, and adiponec in,
48,49
as well as IGF-I,
we e also p edominan in his s udy. Inflamma ion-
FIG. 5. Specific p o ein concen a ions in OR s. labo a o y. Analysis o 16 di e en adipokines in samples
p oduced in labo a o y in op imiza ion phase e sus samples p oduced in OR a e op imiza ion. Adipokines
analyzed we e VEGF, IL-6, adiponec in, lep in, an es, FGFb, esis in, IL-8, MIP-1, TNFa, IFNc, G-CSF, GM-CSF,
IGF-1 and IL-1a. Resul s depic ed as mean –SD, n‡6.
Lopez, e al.; BioResea ch Open Access 2016, 5.1
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275
ela ed ac o s obse ed by o he in es iga o s included
lep in, esis in, adiponec in, TNFa, IL-6, and plasmin-
ogen ac i a o inhibi o -1.
50
In obese indi iduals, he e
is an inc eased exp ession o p o-inflamma o y cy o-
kines such as TNFa, TGFb, and IL-6.
51
In ou p e ious
s udies, FGFb, IGF-I, and IL-6 seemed o sugges he
adipogenic and angiogenic po en ial o ATE.
31,32
VEGF ini ia es angiogenesis,
52
while IGF-1 and FGFb
p omo e wound healing.
53
Howe e , de e mining he
op imal g ow h ac o cock ail o adipogenesis and an-
giogenesis is challenging. The e o e, bo h he o al p o-
ein concen a ion measu emen and a se o g ow h
ac o s measu ed should be kep as a quali y assu ance
me hod also in u he s udies ega ding ATE.
I is he au ho s’ belie ha he main poin o his s udy
was o conse e he g ow h ac o yield when ans e ing
om a labo a o y me hodology o an OR se ing. Va ia-
ions in g ow h ac o concen a ions a e mul i ac o ial,
bu we belie e ha in e pa ien a ia ions play an impo -
an ole. The e o e, we ocused on main aining p o ein
yieldcompa ed op e ious publica ions
31,32
ha demon-
s a ed p o en biological ac i i y.
The p o ocol op imiza ion showed ha ATE is easily
p oduced in he OR wi h a simplified me hod ha p e-
se es s e ili y and lays he ounda ion o implemen
ATE in o he clinical se ing. One o he majo goals
o his de elopmen p ocess was o find accep able
ange o a iabili y o an op imal ATE p epa a ion,
wi hou comp omising he ime p essu e o a su gical
se ing. A dis inc ad an age o ATE is ha i no
only is p epa ed eshly om each pa ien o immedi-
a e use (e.g., ea wounds) bu also ha i can be ozen
and used o sequen ial ea men s. This elimina es he
need o consecu i e su ge ies subs an ially educing
he su geon’s wo kload and also inc easing pa ien
com o .
47
Ma e ial OR equi emen s o ATE p epa-
a ion a e minimal and he need o cen i uga ion o
ac i a ion is bypassed. The ac ha su ficien p o ein
con en can be ob ained in an e en sho e incuba ion
ime han s udied ea lie
32
and unde RT makes ATE a
mo e a ac i e choice o clinicians compa ed o cu -
en ly used p oduc s.
O hobiologics is a ela i ely new science ha in-
ol es applica ion o na u ally ound ma e ials
om biological sou ces (e.g., cell-based he apies)
and o e s exci ing new possibili ies o p omo e and
accele a e bone and so issue healing.
54,55
The
goal o his discipline is o enhance he body’s inna e
abili y o epai and egene a e.
55
In ac , he induc-
i e mic oen i onmen has p e iously shown o en-
hance mig a ion o adipose s omal cells in he
su ounding issue and s imula e he cells o di e en-
ia e in o ma u e adipocy es.
56
G ow h ac o s a e
known o play essen ial oles in wound epai and
may hold he key o success ul healing. Howe e ,
he use o single cy okines has gene a ed disappoin -
ing esul s compa ed o a combina ion o hese.
47,57
The e o e, i is e iden ha wound healing is a e y
complex p ocess ha elies on he ha monious ac ion
o a my iad o g ow h ac o s. This is a undamen al
p inciple in wound healing.
47
The use o sec e o y
ac o s om adipose issue o influence he wound
mic oen i onmen may be a easible app oach o de-
elop opical applica ions o quicke epai .
58
We
sugges he use o adipose issue sec e ome o p o-
duce an ex ac ich in cy okines o opical applica-
ion o wounds, a he han using he di ficul p ocess
o en iching he pa ien s’ s em cells in i o.
58
Bu -
densome s eps, including he use o collagenase o
diges ion o issue o isola e s em cells, would be ob-
ia ed when gene a ing ATE, an impo an ad an-
age in he clinical se ing.
58
To da e, adipose issue
is al eady used as an ac i e bio d essing o ea
wounds wi h p omising wound healing esul s.
58,59
Simila ly, adipose issue and i s sec e ome ha e pos-
i i ely influenced wound healing and issue egene a-
ion.
60,61
In ac , he speed and quali y o wound healing
ha e been enhanced significan ly in wounds ea ed wi h
adipose issue-de i ed ac o s.
58,60
In i o, he condi-
ioned medium o adipose issue composed o a mul i-
ude o adipokines and g ow h ac o s, p o ed o
po en ly induce he p oli e a ion o adipose s em cells
and endo helial cells, compa able o he condi ioned
medium o s em cells.
59,62
The po en ial clinical use o ATE is cu en ly unde
s udy in di e en clinical applica ions whe e g ow h ac-
o may be equi ed o enhance issue ascula iza ion,
healing, cell mig a ion, g ow h, and p oli e a ion. The
use o ATE would g an he benefi s o a cell- ee ap-
p oach and would, he e o e, ha e a mo e ex ended
scope,no jus limi ed oau ologous applica ion.
58
Fu he mo e, i would make he p oduc ion o an o -
he-shel p oduc acco ding o good manu ac u ing p ac-
ice much easie . Especially o he po en ial clinical appli-
ca ions o adipose issue, he e ec o i s sec e ome needs
o be in es iga ed ho oughly.
58
In addi ion, he p oan-
giogenic e ec epo ed in his s udy may pose as an a -
ac i e s a ing poin o in es iga ing he po en ial
impac o adipose issue sec e o y ac o s on neo ascula-
iza ion in se e al issue egene a ion applica ions.
Lopez, e al.; BioResea ch Open Access 2016, 5.1
h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030
276
Conclusion
ATE is a e y p omising bioac i e agen o a ple ho a o
clinical uses. The ela i e ease o p epa a ion, applicabili y
in he clinical se ing, a o able sa e y p ofile, and possible
beneficial ou come make ATE a p omising he apeu ic
app oach o u u e egene a i e ea men s. I is easy o
ob ain, inexpensi e, and by being au ologous and cell-
ee, i is minimizing common p oblems o issue engi-
nee ing. I s p o en adipogenic and angiogenic p ope ies,
along wi h being an abundan sou ce o g ow h ac o s,
make ATE an appealing mic oen i onmen o cell p o-
li e a ion, mig a ion, and di e en ia ion. Unde hese ci -
cums ances, any clinical scena io in need o issue olume
addi ion, wound healing, and adequa e ascula iza ion
would benefi om ATE. The consis en p o ein and
g ow h ac o concen a ions p o e he epea abili y o
he me hod, and hus, he ex ac p oduc ion me hod
can be success ully p oduced in he OR en i onmen
and is now eady o clinical esea ch.
Acknowledgmen s
We hank he heal hca e s a a Tampe e Uni e si y
Hospi al, as well as he pa ien s, o hei collabo a ion
conce ning adipose issue sample dona ions. The au-
ho s acknowledge Ris o Vuen o, MD, PhD, and he
s a a Fimlab Labo a o ies Oy o conduc ing he s e-
ili y es ing on he samples. A he Uni e si y o Tam-
pe e, he au ho s hank Ms. Sa i Leinonen and Ms.
Hilkka Ma¨kinen o hei excellen echnical assis ance.
This s udy was financially suppo ed by he Compe i-
i e S a e Resea ch Financing o he Expe Responsi-
bili y a ea o Tampe e Uni e si y Hospi al, g an
9R025, O ion-Fa mos esea ch ounda ion, and he
Finnish Funding Agency o Technology and Inno a-
ion (TEKES). Addi ional unding o he p ojec was
p o ided by The Diabe es Resea ch Founda ion.
Au ho Disclosu e S a emen
Pa en pending WO2010026299A1.
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