Cytokine-Rich Adipose Tissue Extract Production from Water-Assisted Lipoaspirate: Methodology for Clinical Use

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 Lopez, e al.; BioResea ch Open Access 2016, 5.1 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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. Lopez, e al.; BioResea ch Open Access 2016, 5.1 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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. Lopez, e al.; BioResea ch Open Access 2016, 5.1 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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. Lopez, e al.; BioResea ch Open Access 2016, 5.1 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 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. Re e ences 1. Cla k RA, Ghosh K, Tonnesen MG. Tissue enginee ing o cu aneous wounds. J In es De ma ol. 2007;127:1018–1029. 2. Elçin YM, Dixi V, Gi nick G. 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Lopez, e al.; BioResea ch Open Access 2016, 5.1 h p://online.liebe pub.com/doi/10.1089/bio es.2016.0030 277