Research Article Human Adipose-Derived...

Research ArticleHuman Adipose-Derived Mesenchymal Stem CellsCryopreservation and Thawing Decrease 1205724-Integrin Expression

Ana Carolina Irioda12 Rafael Cassilha3 Larissa Zocche1

Julio Cesar Francisco1 Ricardo Correa Cunha1 Priscila Elias Ferreira1

Luiz Cesar Guarita-Souza4 Reginaldo Justino Ferreira1 Bassam Felipe Mogharbel1

Venkata Naga Srikanth Garikipati5 Daiany Souza1 Mirian Perlingeiro Beltrame6

and Katherine Athayde Teixeira de Carvalho12

1Cell Therapy and Biotechnology in Regenerative Medicine Research Group Pele Pequeno Prıncipe InstituteAvenida Silva Jardim 1632 80250-200 Curitiba PR Brazil2Bioprocess Engineering and Biotechnology Department Federal University of Parana Avenida Coronel Francisco Heraclito dosSantos 210 81531-970 Curitiba PR Brazil3Hospital Sante Curitiba Plastic Surgery Clinic Rua XV de Novembro 2913 80045-340 Curitiba PR Brazil4Experimental Laboratory of Institute of Biological and Health Sciences of Pontifical Catholic University of Parana (PUCPR)Rua Imaculada Conceicao 1155 80215-901 Curitiba PR Brazil5Center for Translational Medicine Temple University School of Medicine Philadelphia PA 19140 USA6Flow Cytometric Analysis Laboratory Clinical Hospital of Federal University of Parana Rua Padre Camargo 280Alto da Gloria 80060-240 Curitiba PR Brazil

Correspondence should be addressed to Katherine Athayde Teixeira de Carvalho katherinecarvgmailcom

Received 16 October 2015 Revised 5 December 2015 Accepted 15 December 2015

Academic Editor Hiroshi Mizuno

Copyright copy 2016 Ana Carolina Irioda et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aim The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented as there is agrowing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies The aimof this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34 CD45 CD73CD90 CD105 and CD49d) colony forming unit ability viability and differentiation potential before and after cryopreservationMaterials andMethods 12 samples of the adipose tissuewere collected from ahealthy donor using the liposuction techniqueThe cellisolation was performed by enzymatic digestion and then the cells were cultured up to passage 2 Before and after cryopreservationthe immunophenotype cellular viability analysis by flow cytometer colony forming units ability differentiation potential intoadipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining respectively Results The immunophenotypicmarkers expression was largely preserved and their multipotency was maintained However after cryopreservation the cellsdecreased 1205724-integrin expression (CD49d) cell viability and number of colony forming units Conclusions These findings suggestthat ADMSC transplanted after cryopreservationmight compromise the retention of transplanted cells in the host tissueThereforefurther studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy

1 Introduction

In the development of new therapies using stem cellsmesenchymal stem cells (MSC) from diverse origins haveexhibited immense potential [1 2] Although the reportsrelated to the therapeutic potential of MSC most of them

are performed mainly on hematopoietic origin more frombonemarrow (BM) that is explained by the extensive clinicalexperiments with stem cells particularly in the treatment ofoncohematological diseases [3 4] However adipose tissue-derived mesenchymal stem cells (ADMSC) emerged as apromising candidate As ADMSC are adult stem cells that

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 2562718 9 pageshttpdxdoiorg10115520162562718

2 Stem Cells International

can be isolated easily from adipose tissue the cells areobtained through plastic surgery that represents an ampleand accessible source of adult stem cells with the ability todifferentiate into adipocytes osteocytes chondrocytes andcells phenotypically similar to neurons [5] Due to ADMSCability to differentiate into several cell types of clinical inter-est they hold an immense potential for the future therapeuticuse of these cells in clinics Although many reports relatedto the therapeutic potential of ADMSC most of the currentinvestigationswere performedmainly on freshly isolated cells[6] As this cell type holds a clinical translation value there isan increasing interest in stem cell banking of ADMSC

In consequence of the inherent characteristics of theADMSC low immunogenicity higher cellular yield incomparison to bone marrow cells and pluripotency thecryopreservation of these cells would be a very convenientalternative However the effects of the cryopreservationand the thawing proceedings could impact their therapeuticoutcome and therefore should be evaluated Once the cellsare cultivated and are attached to the plastic before thecells cryopreservation these are subjected to a subtractdesegregation stress being able to put in risk the integrityof the cell membrane and its surface molecules for the celladhesion After thawing the cryopreserved cells have also therisk of injuring the membrane and other cellular functionalcharacteristics both proceedings could affect the therapeuticoutcomes

In this context the present study aims to address whetherthe ADMSC cryopreservation could compromise the cellintegrity the expression of adhesion molecules andor thedifferentiation potential of ADMSC and other stem cellslineages

2 Material and Methods

21 Experimental Design Adipose-derived mesenchymalstem cells were obtained from adipose tissues of 12 adulthealthy donors (10 female and two male) by liposuctionperformed by a plastic surgeon with informed consentsignature from each patientThe cell isolation was performedby enzymatic digestion with collagenase type I Then thecells were cultured in DMENF12 supplemented with 10 ofcalf fetal serum 100 unitsmL of penicillin and 100 120583gmLof streptomycin Before and after cryopreservation thefollowing assays were performed colony forming unitsimmunophenotype and cellular viability with Annexin Vand 7-AAD analysis by flow cytometer as well as ADMSCdifferentiation to adipocytes and osteoblasts

22 Isolation of ADMSC The adipose tissue samples werecollected from a healthy donor after consent signature fromeach patient All the samples were collected from specializedplastic clinics using the liposuction technique After thesamples were collected they were cultured before 24 hoursusing the following protocol 50mL of each sample wasextensively washed with phosphate buffered saline (PBS)(Sigma St Louis MO USA) containing 300 unitsmL ofpenicillin and 300 120583gmL of streptomycin (Sigma St Louis

MO USA) (PBSPS) After washing the samples and debrisremoval these were placed in 50mL tubes with 0075 oftype I A collagenase (Sigma St Louis MO USA) preparedin PBSPS The cells were homogenized and incubated withagitation at 37∘C for 30 minutes After the incubation thetype I collagenase activity was ceased by adding the samevolume of Eagle modified from DulbeccoF12 (DMEMF12)(LGC Biotechnology Brazil) containing 10 of fetal calfserum (FCS) (GIBCO B Life Technologies Inc RockvilleUSA RL) as determined to be standard culture medium(SCM) Then the samples were centrifuged at 400 g for 10minutes The supernatant was discarded and the pellet wasresuspended in 10mLof PBSPS then another centrifugationwas made at 400 g for 10 minutes The supernatant wasdiscarded and the pellet was suspended in 5mL of culturemedium The cell suspension was filtered in cell strainer of100 120583M(BectonDickinsonUSA)The cell countwasmade bya hemocytometer and the utilization of Trypan Blue (SigmaSt Louis MO USA) (for the dilution of 1 1 10 120583L of the cellsuspension was added for 10 120583L of Trypan Blue) After that1 times 105 cells for cm2 in 25 cm2 and 75 cm2 culture flasks wereharvested and incubated at 37∘C and 5 of CO

2[7ndash9]

23 Cultivation and Expansion 72 hours after on cultivationthe medium was aspirated from the flasks and the cellswere washed with preheated PBSPS then 7mL of culturemedium was added in the 25 cm2 and 12mL in the 75 cm2flasks The cells were kept incubated at 37∘C and 5 CO

2

Themediumwas changed twice a week until the cells reachedbetween 80 and 90 of confluence After the desiredconfluence was reached the cells were briefly washed withpreheated PBSPS and added trypsinEDTA (025) (SigmaSt Louis MO USA) for disaggregation of the adherent cellsThe cells were incubated for 10 minutes at 37∘C Then thesame volume of SCM was added for the neutralization of thetrypsin action The cell suspension was transferred to sterilecentrifuge tube and centrifuged at 400 g for 10 minutes Thesupernatant was discarded and the cells were resuspended inapproximately 5mL of SCMThe cell count was realized witha hemocytometer chamber using Trypan Blue (1 1 dilution)[9] After the counting cells were once again harvested in75 cm2 flasks at a concentration of 1 times 103 cellscm2 andincubated at 37∘C and 5 of CO

2until they reached 80 of

confluence When they reached the desired confluence thecells were trypsinized andwere available for analysis For eachsample the culture was done at passage 2 (P2)

24 Cryopreservation Proceedings After trypsinization at P2cells as described previously and confirming their viabilityby the Trypan Blue exclusion method a fraction of the cellswere submitted to the cryopreservation proceedings Thecells were counted with a hemocytometer (approximately1 times 106 cellsmL) The cryoprotectant medium had 80 ofBFS 10 of dimethyl sulfoxide (DMSO) (Sigma St LouisMO USA) 10 of DMEM-F12 (LGC Biotechnology Brazil)and 100 unitsmLof penicillin and 100120583gmLof streptomycin(Sigma St Louis MO USA) 1mL aliquots (containing 1 times106 cells) were transferred to preidentified cryogenics tubes

Stem Cells International 3

Table 1 Panel of monoclonal antibodies used in this study for immunophenotypic characterization

Marker Clone Reactivity Fluorochrome Function

CD34 581CD34 Human FITC

Stem cell marker (precursor) also foundin hematopoietic progenitor cellsvascular endothelium and fibroblasts ofthe same tissue Probably it works as atransduction signer and has a function inendothelium specific antigen adhesion

CD45 (common leukocyteantigen) HI30 Human PE

Leukocytes marker CD45 proteins arelocated in all hematopoietic cells but notin erythrocytes

CD49d (integrin 1205724) 9F10 Human PETransmembrane glycoprotein integrin1205724 It makes several cell-cell interactionand cell-matrix and participated incellular adhesion

CD73(ecto-51015840-nucleotidase) AD2 Human PE

It have been suggested that this markercan mediate costimulator signals in thecells T activation and endothelium as wellcatalyses the dephosphorylation ofadenosine monophosphate in adenosine

CD90 (THy-1) 5E10 Human FITC

The role of interaction in cell-cell andcell-matrix has been speculated withrelated neurites growth nerveregeneration apoptosis metaphaseinflammation and fibrosis

CD105 (endoglin) 266 Human Purified Responsiveness modulator of TGF-120573cellular complex

(cell type density and date) The tubes were cooled in aprogrammable freezing device (Nicool LM10 Air LiquideMarne La Vallee France) The cryopreservation equipmentstarts at room temperature and in the end of the first stage(Program 3ndash15 minutes) reaches the temperature minus30∘C inthe end of step 2 (Program 5ndash45 minutes) the temperature isminus60∘C finally in the last stage step 3 (Program9-10minutes)the temperature is minus110∘C After step 3 the cells were trans-ferred to the liquid nitrogen to minus196∘CThis method preventsthe crystals formation inside the cell because the freezingoccurs slowly [9ndash11] Shortly after the tubes were transferredto the liquid nitrogen where they remained stocked forapproximately 20 days All the samples were cryopreservedin triplicate

25 Thawing Proceedings 20 days after the cryopreservedcells were thawed at 37∘C and transferred in 5mL of SCMand gently homogenizedThe cell suspension was transferredfor sterile centrifuge tube and centrifuged at 400 g for 10minutes The supernatant was discarded and the cells wereresuspended in approximately 5mL of culture medium Thecells were submitted to analysis

26 Colony Forming Units (CFU) Analysis Before and afterthe cryopreservation 10 cells per cm2 were seeded intriplicate in wells of 9 cm2 with SCM the medium waschanged according to other steps of culture After 14 daysthe medium was removed and the cells were fixated withmethanol for 5 minutes and then stained with 05 of crystal

violet inmethanol formore 5minutesTheflaskswerewashedtwice with PBSPS and dried The number of colonies withmore than 2mm was counted and the results represent thenumber of former colonies by 100 seeded cells (countedcoloniesinoculated cells) times 100 [12]

27 Immunophenotypic Analysis All samples were analyzedfor the expression of surface markers (Table 1) using mono-clonal antibodies against cluster of differentiation (CD) anti-gens conjugated with fluorochromes and analyzed beforethe cryopreservation and after thawing by flow cytometerCells (number) were incubated with the antibodies that aredescribed in Table 1 and corresponding isotypes were usedThe primary antibodies were incubated for 10 minutes in thedark The antibody against CD105 is a purified antibody andit is not conjugated with fluorescence for this reason it wasnecessary to incubate it with a secondary antibody for more15 minutes in the dark room and redo the cell washing stepFurther 5 120583L of 7-AAD was added and incubated in the darkroom for 15 minutes After that 400 120583L of binding bufferwas added and the samples were analyzed by flow cytometer(FACS Calibur Becton Dickinson San Diego USA) [11 1314] Prior to each test the equipment was calibrated usingBDCalibrite (Becton Dickinson San Diego USA) accordingto the manufacturerrsquos instructions All antibodies were usedaccording to the manufacturerrsquos instructions 20000 events(cells) were captured the percentage values of each markerwere analyzed through specific analysis software Cyflogicversion 121


Table 2 Surface markers expressions before cryopreservation and after thawing

Molecular marker CD34minus CD34+ CD45minus CD49d+ CD73+ CD90+ CD105+Cryopreservation Before After Before After Before After Before After Before After Before After Before AfterMedium 9888 993 112 078 9979 998 8867 778 9957 995 9955 995 994 983Max 9986 100 332 21 100 100 9638 931 9994 999 9997 100 9991 999Min 9698 979 014 003 9927 996 8015 416 9851 985 9815 975 9787 939SD 108 061 108 06 022 014 655 145 043 043 007 07 064 207119901 value 0113 0158 0791 0007lowast 0528 0618 005lowast

119901 lt 005

28 Cell Viability with Annexin V and 7-AAD AnalysisThe adherent cells were detached with trypsin (025)centrifuged for 3 minutes at 400 g and resuspended in 2mLof PBS After that in 5mL tubes 100 120583L of this solution wasadded After that 400 120583L of PBS was added in each tubethey were homogenized and centrifuged The supernatantwas discarded and 100120583L of the binding buffer was addedAfterwards 5 120583L of Annexin V was added 5 120583L of 7-AADwas added and the tubes were incubated in the dark roomfor 15 minutes After incubation 400 120583L of binding bufferwas added and homogenized the data was collected by flowcytometer (FACS Calibur Becton Dickinson USA) [11]

29 Adipogenic Differentiation The cells were treated withadipogenic medium consisting of DMEM-F12 mediumcontaining 10 FBS 05m M isobutylmethylxanthine(Sigma St Louis MO USA) 1mmolL dexamethasone(Sigma St Louis MO USA) 10mgmL insulin and 50 120583Mindomethacin (Sigma St Louis MO USA) and wereincubated at 37∘C and 5 CO

2 Control cells were treated

with SCM Medium was changed two times a week 14 daysafter the cells were fixed and stained with Oil Red Stain(Sigma St Louis MO USA) to demonstrate the fat dropletsin the cells [7 8]

210 Osteogenic Differentiation The osteogenesis was in-duced by keeping the cells in DMEM-F12 medium with100 unitsmL of penicillin 100120583gmL of streptomycin 1 nMof dexamethasone 2mM of 120573-glycerophosphate (SigmaUSA) and 50 120583M of ascorbate-2-phosphate (Sigma USA)and incubated at 37∘C and 5CO


with SCM The cells were kept in these conditions for 35days and the medium was changed twice per week Theexperimental and control cells were fixed glutaraldehyde at25 for ninety minutes and stained with Alizarin Red Stain(Sigma St Louis MO USA) to demonstrate mineralization[7 8]

211 Statistic Analysis The obtained results in this studywere expressed as mean plusmn standard deviation (SD) Forthe comparison between the molecules expression analysisbefore and after cryopreservation the 119905-test was performed119901 lt 005 was considered significantThe data were organizedin Excel spreadsheet andwere analyzedwith Statistica version90 software

CD34minus CD45minus CD49d+ CD73+ CD90+ CD105+Markers

Before cryopreservationAfter thawing

lowastlowast

50

60

70

80

90

100

110

Expr

essio

n (

)

Figure 1 Surface markers expression of cells before cryopreserva-tion and after thawing The analysis was done by Cyflogic software121 The 119905-test was done 119901 lt 005 was considered significant Theresults were 119901lowastlowast = 0 001

3 Results

31 Phenotypic Analysis of ADMSC Flow cytometric analysisshowed a typical mesenchymal phenotype of ADMSC withexpression of CD73 CD90 and CD105 whereas these cellslacked expression of CD34 and CD45 Interestingly weobserved a significant reduction of CD49d expression afterthawing cryopreserved ADMSC (Table 2 Figures 1 and 2)

32 Annexin V 7-AAD Staining The differences in CD49dexpression before and after cryopreservation led us to look atthe cell viability before and after cryopreservation Cell viabil-ity was assessed by Annexin V 7-AAD staining we observeda significant reduction in viability from 9134 plusmn 454 to7499 plusmn 1419 (119901 = 0001) after cryopreservation losingan average of 179 viable cells Concerning labeling withAnnexin V (apoptosis) values were very close to the values ofcellular viability being 9139 plusmn 55 before cryopreserva-tion and 7631plusmn 1333 after thawing (119901 = 0003) (Table 3Figure 3) Thus suggesting that the majority of Annexin Vstained cells were also stained with 7-AAD which means thatthe amount of cells only in apoptosis was a small proportion

33 Colony Formation Assay Further we looked at thecolony formation ability of ADMSC and observed a sig-nificant decrease in the colonies formation capacity CFUs


Before BeforeAfter After

CD34 CD34 CD73 CD73

CD45 CD45 CD90 CD90

CD49d CD49d CD105 CD105

Figure 2 Histograms of ADMSC markers before and after cryopreservation The grey color represents specific marker and the white colorrepresents an isotype control

Before After

Annexin V Annexin V

7-AAD 7-AAD

Figure 3 Histograms of Annexin V (apoptosis marker) and 7-AAD(viability marker) of the cells before and after cryopreservationThegrey color represents specific marker and the white color representsan isotype control

before and after cryopreservation were 2808 plusmn 706 ver-sus 2151 plusmn 661 (119901 lt 001)

34 Adipogenic Potential of ADMSC It was assessed aftercryopreservation with a lineage-specific induction medium

the cells differentiated into adipogenic as evidenced by OilRed whereas control cells did not take up Oil Red Staining(Figure 4)

35 Osteogenic Potential of ADMSC In addition upon treat-ment with a lineage-specific induction medium the cellsdifferentiated into osteogenic as evidenced by Alizarin Redwhereas control cells did not take up Alizarin Red staining(Figure 5)

4 Discussion

The ADMSC are cells with a huge therapeutic potential andimportant tool to cellular metabolism studies Therefore thecharacterization of cryopreserved cells and theirmaintenanceafter thawing cryopreserved are relevant

About the CFU analysis we observed a significantdecrease in the colonies formation capacity CFUs beforeand after cryopreservation were 2808 plusmn 706 versus2151 plusmn 661 (119901 = 001) respectively These results arein agreement with the results found by Goh and colleagues(2007) that cryopreservation causes decrease in adhesionefficiency of ADMSC [15] This difference could be relatedto decreased expression of integrin 1205724 (CD49d) that wehave observed Mitchell et al (2006) reported that the abilityto form colonies of ADMSC is considerably greater thanBMSC even after cryopreservationThis group also reportedthat the more the passages the greater the number of CFUs


(a) (b)

Figure 4 Adipose differentiated cells after 14 days in induction medium sample after thawing of cryopreserved cells phase contrastmicroscopy 250x (a) Presence of fat droplets (stained with Oil Red) in ADMSC cultivated with adipogenic induction medium (b) Controldoes not have fat droplets indicating the undifferentiated cells cultivated with standard medium Scale (10 120583m)

(a) (b)

Figure 5 Osteogenic differentiated cells after 35 days in osteogenic induction medium sample after thawing of cryopreserved cells 250xrise (a) ADMSC stained with Alizarin Red demonstrating the presence of calcium in extracellular matrix (b) Control without matrixdemonstration indicating the undifferentiated cells cultivated with standard culture medium Scale (10120583m)

and suggests that the passages select a particular cell typepossibly the stem cells [16] The same parameters werekept before and after cryopreservation and however canindeed be a delay in cell growth considering the fact thatthe cells were again adapting to cultivation conditions andthe limited size of the colonies Regarding cellular therapythe increase of number of the passages could compromisethe safety and that is why it may be related to increasingmutations and increasing the transformation of the cells[17] On the other hand about the differentiation processmost of the groups studying with ADMSC report that after14 days of adipogenic induction all samples of cells have amorphology consistent with adipocyte which was confirmedby staining of lipids with Oil Red as well as by PCR thatidentify genes consistent with adipocytes and which are notpresent in undifferentiated cells Rodriguez and colleagues(2004) reported that after this period about 90 of the cellsaccumulate intracellular lipid and that there might also be nosignificant accumulation in untreated cells [18] All samples

before and after thawing that were induced to the adipogenicdifferentiation medium stained positive for Oil Red showingthe presence of lipids inside the cells (Figure 4(a)) In thecontrol medium the standard culture medium was addedand there was no trace of lipids indicating that these cellsdid not differentiate into adipocytes (Figure 4(b)) Theseresults clearly show that ADMSC have after appropriateinducing an adipogenic differentiation capacity To identifyosteogenic differentiation several groups have reported thatbetween 14 and 21 days after induction we can observe thepresence of a mineralized matrix by the calcium phosphatestaining with Alizarin Red [16 19] In the present work itwas observed only after 30 days in the induction mediumAll samples before and after thawing that were inducedto the osteogenic differentiation medium stained positivefor Alizarin Red (Figure 5(a)) In the control medium thestandard culture medium was added and there was noevidence of mineralization indicating that these cells didnot differentiate into osteogenic differentiation (Figure 5(b))


Both adipogenic and osteogenic differentiation resultssuggest that cells were stem cells Other researchers reportedthe maintenance of the capacity of differentiation of ADMSCin both adipocytes and osteocytes after thawing [19ndash21] Inthis study 100 of the samples had the same induction time

There are some controversies about the markers forMSC This occurs because of different designs of differenttypes of markers however there is a consensus that MSCare negative for CD45 (a marker of HSC) [22] In thisstudy all samples were negative for CD45 which is anindication that they would be large MSC In this study foursamples showed a small population of cells positive for CD34(1 to 3)

In this study varying values for the CD49d were found(8867 plusmn 655) consistent with the values found by Katz andcolleagues (2005) (78 plusmn 20) [23] Maybe if those cells willbe submitting culture again it will be possible to recoverthis expression of CD49d In this work cultivating thecells after cryopreservation was not possible due to thelow number of cells However thinking about translationto humans the most likely hypothesis in a cell therapywould be the immediate transplant of the cells after thawingThus as already described in the literature increasing thenumber of passages also increases the probability of celltransformations such teratomes which makes impracticabletherapy [18] In addition to varying values of expression thissurface marker showed significant decreases after thawingof cryopreserved cells (778 plusmn 1445 119901 = 0007) Thismarker represents the 1205724-integrin an adhesionmolecule thatinteracts with the 1205731 integrin forming a heterodimer lateactivating antigen-4 (VLA-4) [13] Lei and colleagues (2007)found low positive values for CD49d (average 126) but theway of isolation of ADMSC was through the subcutaneoustissue and not through lipoaspirate another issue is that theyused DMEM with low glucose these differences protocolscan select different types of cells with similar characteristicsbut not identical [24] For Katz and coworkers (2005) somedifferences between groups with respect to expression ofCD49d reflect adjustments of countless variations of thesecells to the extracellular medium such as density cellcycle culture time and the number of passages [23] Theenvironment produces appropriate niches for stem cells andregulates the maintenance of these niches for specific celllines For this regulation the adhesion of stem cells in theextracellular matrix is critical as it allows communicationbetween the cells and the matrix being a prerequisite for themaintenance of tissue [25]Thus these changes in expressionof CD49d after cryopreservation may mean a major problemin transplantation of these cells causing them not properlycommunicating with the injured tissue

In this study only viable and intact cells were ana-lyzed Gonda et al (2008) stated that cryopreservation cancause structural and functional damage to cell proteinsand reduce their viability but immunophenotypic exchangescould hardly occur [19] Expression changes really wouldnot have excuses but the loss of expression is very relevantbecause the group itself mentioned that cryopreservation cancause damage to the membrane protein which is the case ofCD49d which represents an adhesion protein Few studies

Table 3 Representation of viability and integrity cells beforecryopreservation and after thawing

Annexin V 7-AADBefore After Before After

Media 9139 7631 9134 7499Max 962 9583 7929 4921Min 7518 5238 9517 9527DP 585 1333 454 1419119901 value 0003 0001

are related to immunophenotypic difference after thawingof cryopreserved cells which is an extremely importantfeature that should be studied [19 26] Cellular viabilitiesbefore cryopreservation and after thawing were analyzed bystaining with the kit of viability (BD) Annexin V PE 7-AADon 20000 events Apoptosis of the cells is characterizedby phosphatidylserine a component of the inner leafletof cell membranes When a cell enters apoptosis processphosphatidylserine becomes exposed on the outer wall ofthe membrane but the cell membrane remains intact Thecells positive for Annexin V represent cells that have thistranslocation of phosphatidylserine [27] After thawing therewas a significant loss of the cells integrity being 165lower than the cells before the cryopreservation (Figure 5)Maybe that cell integrity is also lost with thawing Anothercells line which was positive for 7-AAD indicates thatthe membrane integrity was compromised consequentlythere was cell death Accordingly these markers are used todistinguish dead cells from cells that are in the process ofapoptosis To analyze these markers Cyflogic software 121was used which provides the histogram overlay of the isotypecontrol with the results of fluorescence of each marker andquantifies the percentage of positive (not overlapping theisotype control) and negative (superimposed on the isotypecontrol) markings Thus it was possible to analyze threevariables (i) 100 viable cells (negative for Annexin V-PEand negative for 7-AAD) (ii) dead cells (positive for AnnexinV-PE and positive for 7-AAD) and (iii) the process of apop-tosis in cells (positive for Annexin V-PE and negative for 7-AAD) Following these parameters before cryopreservationviability was 9134 plusmn 454 After thawing the cells hada significant drop in cell viability 7499 plusmn 1419 (119901 =0001) losing on average 179 viable cells Concerninglabeling with Annexin V (apoptosis) values were very closeto the values of cellular viability being 9139 plusmn 55 beforecryopreservation and 763plusmn 1333 after thawing (119901 =0003) (Table 3) This study demonstrates that the majorityof Annexin V stained cells were also stained with 7-AADwhich means that the amount of cell only in apoptosis wassmall

The ADMSC viabilities of cryopreserved cells after thaw-ing may be explained with the concentration of cells in eachcryotube Goh et al (2007) tested four cell concentrations25 times 105 5 times 105 1 times 106 and 2 times 106 per mL and founda viability of 714 8110 779 and 692 respectivelyIn this study the cryopreservation of cells in 1 times 106 cells


per mL and viability found values similar to values found byGoh group (2007) however the method used by Goh et al(2007) was staining by Trypan Blue which is more relativeto be counted manually the method used in this study ismore accurate by flow cytometric analysis [15] Thirumalaand colleagues (2010) found viabilities staying at 84 plusmn 8when using the same cryoprotectant in their study but thetest was performed on P1 [27] De Rose and colleagues (2009)found amazing values of cellular viability 925 This highrate of viability may be related to the form of thawing thesecells which were transferred to culture medium with 10FCS prior to complete thawing it could be explained by thefact that the cells stayed less time in contact with DMSOin room temperature that is known for its cytotoxic effects[10] The researchers have shown that many factors influencethe intracellular dynamics when cells are frozen affecting theviability of these cells Among these factors can be highlightedthe formation of intracellular ice which can perforate thecell membranes and high concentration of cells which canlimit the space preventing cell growth during freezing [15] Astandard protocol was used for various cell types but somecells have specific characteristics which may require specialcare For this reason it would be ideal to develop a specificprotocol for ADMSC to improve the viability rate and othercharacteristics [11 19 28]

Some authors used as long-term storage method freezingin a freezer at minus80∘C rather than liquid nitrogen (minus196∘C)but this method has revealed lower levels of viability whileretaining the functional characteristics Other variables couldinfluence the viability like the speed of freezing because if itwill be faster there are greater probabilities of intracellularice formation and consequent membrane damage [19 26]and the choice of serum free media which may not benefitfrom a large cell viability However considering therapeuticapplications even with this low viability could be indicatedby the reduced risk of contaminating [26]

5 Conclusions

In this study the standard protocols of the human adipose-derived mesenchymal stem cells cryopreservation and thaw-ing proceedings are demonstrating the decrease 1205724-integrinexpression (CD49d) cell viability and colony forming unitsafter thawingThese findings can compromise the integrationof cells in the extracellular matrix of the host tissue Furtherprotocols should be established to improve and ensure the cellgraft

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are thankful to Coordination for the Improve-ment of Higher Education Personnel (CAPES) for the finan-cial support

References

[1] N Kim and S-G Cho ldquoClinical applications of mesenchymalstem cellsrdquo Korean Journal of Internal Medicine vol 28 no 4pp 387ndash402 2013

[2] X Wei X Yang Z-P Han F-F Qu L Shao and Y-F ShildquoMesenchymal stem cells a new trend for cell therapyrdquo ActaPharmacologica Sinica vol 34 no 6 pp 747ndash754 2013

[3] M Ogawa ldquoDifferentiation and proliferation of hematopoieticstem cellsrdquo Blood vol 81 no 11 pp 2844ndash2853 1993

[4] B Logan E Leifer C Bredeson et al ldquoUse of biologicalassignment in hematopoietic stem cell transplantation clinicaltrialsrdquo Clinical Trials vol 5 no 6 pp 607ndash616 2008

[5] F De Francesco G Ricci F DrsquoAndrea G F Nicoletti and G AFerraro ldquoHuman adipose stem cells from bench to bed-siderdquoTissue Engineering Part B Reviews vol 21 no 6 pp 572ndash5842015

[6] I A Panfilov R Jong S Takashima and H J DuckersldquoClinical study using adipose-derived mesenchymal-like stemcells in acutemyocardial infarction andheart failurerdquo inCellularCardiomyoplasty vol 1036 ofMethods in Molecular Biology pp207ndash212 Humana Press 2013

[7] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering vol 7 no 2 pp 211ndash228 2001

[8] P A Zuk M Zhu P Ashjian et al ldquoHuman adipose tissue is asource of multipotent stem cellsrdquoMolecular Biology of the Cellvol 13 no 12 pp 4279ndash4295 2002

[9] B A Bunnell M Flaat C Gagliardi B Patel and C RipollldquoAdipose-derived stem cells isolation expansion and differen-tiationrdquoMethods vol 45 no 2 pp 115ndash120 2008

[10] A De Rosa F De Francesco V Tirino et al ldquoA new methodfor cryopreserving adipose-derived stem cells an attractive andsuitable large-scale and long-term cell banking technologyrdquoTissue Engineering - Part C Methods vol 15 no 4 pp 659ndash6672009

[11] K A T Carvalho C C Cury L Oliveira et al ldquoEvaluation ofbone marrow mesenchymal stem cell standard cryopreserva-tion procedure efficiencyrdquo Transplantation Proceedings vol 40no 3 pp 839ndash841 2008

[12] P A Sotiropoulou S A Perez M Salagianni C N Baxevanisand M Papamichail ldquoCharacterization of the optimal cultureconditions for clinical scale production of humanmesenchymalstem cellsrdquo Stem Cells vol 24 no 2 pp 462ndash471 2006

[13] D A De Ugarte Z Alfonso P A Zuk et al ldquoDifferentialexpression of stem cell mobilization-associated molecules onmulti-lineage cells from adipose tissue and bone marrowrdquoImmunology Letters vol 89 no 2-3 pp 267ndash270 2003

[14] K A T Carvalho R B Simeoni L C Guarita-Souza et alldquoAngiogenesis without functional outcome after mononuclearstem cell transplant in a doxorubicin-induced dilated myocar-diopathy murine modelrdquo The International Journal of ArtificialOrgans vol 31 no 5 pp 431ndash438 2008

[15] B C Goh S Thirumala G Kilroy R V Devireddy andJ M Gimble ldquoCryopreservation characteristics of adipose-derived stem cells maintenance of differentiation potentialand viabilityrdquo Journal of Tissue Engineering and RegenerativeMedicine vol 1 no 4 pp 322ndash324 2007

[16] J B Mitchell K Mcintosh S Zvonic et al ldquoImmunophenotypeof human adipose-derived cells temporal changes in stromal-associated and stem cellndashassociated markersrdquo STEM CELLSvol 24 no 2 pp 376ndash385 2006


[17] R J Ferreira A C Irioda R C Cunha et al ldquoControversiesabout the chromosomal stability of cultivated mesenchymalstem cells their clinical use is it saferdquo Current Stem CellResearch andTherapy vol 7 no 5 pp 356ndash363 2012

[18] A-M Rodriguez C Elabd F Delteil et al ldquoAdipocyte differ-entiation of multipotent cells established from human adiposetissuerdquo Biochemical and Biophysical Research Communicationsvol 315 no 2 pp 255ndash263 2004

[19] K Gonda T Shigeura T Sato et al ldquoPreserved proliferativecapacity andmultipotency of human adipose-derived stem cellsafter long-term cryopreservationrdquo Plastic and ReconstructiveSurgery vol 121 no 2 pp 401ndash410 2008

[20] K Oishi H Noguchi H Yukawa et al ldquoCryopreservationof mouse adipose tissue-derived stemprogenitor cellsrdquo CellTransplantation vol 17 no 1-2 pp 35ndash41 2008

[21] G Minonzio M Corazza L Mariotta et al ldquoFrozen adipose-derived mesenchymal stem cells maintain high capability togrow and differentiaterdquo Cryobiology vol 69 no 2 pp 211ndash2162014

[22] C M Kolf E Cho and R S Tuan ldquoMesenchymal stromal cellsBiology of adult mesenchymal stem cells regulation of nicheself-renewal and differentiationrdquo Arthritis Research amp Therapyvol 9 no 1 article 204 2007

[23] A J Katz A Tholpady S S Tholpady H Shang and RC Ogle ldquoCell surface and transcriptional characterizationof human adipose-derived adherent stromal (hADAS) cellsrdquoSTEM CELLS vol 23 no 3 pp 412ndash423 2005

[24] L Lei W Liao P Sheng M Fu A He and G HuangldquoBiological character of human adipose-derived adult stem cellsand influence of donor age on cell replication in culturerdquo Sciencein China Series C Life Sciences vol 50 no 3 pp 320ndash328 2007

[25] F Djouad B Delorme M Maurice et al ldquoMicroenvironmen-tal changes during differentiation of mesenchymal stem cellstowards chondrocytesrdquo Arthritis Research ampTherapy vol 9 no2 article R33 2007

[26] S Mieno R T Clements M Boodhwani et al ldquoCharacteristicsand function of cryopreserved bone marrow-derived endothe-lial progenitor cellsrdquo Annals of Thoracic Surgery vol 85 no 4pp 1361ndash1366 2008

[27] SThirumala J M Gimble and R V Devireddy ldquoEvaluation ofmethylcellulose and dimethyl sulfoxide as the cryoprotectantsin a serum-free freezingmedia for cryopreservation of adipose-derived adult stem cellsrdquo Stem Cells and Development vol 19no 4 pp 513ndash522 2010

[28] M L Gonzalez-Fernandez S Perez-Castrillo P Ordas-Fernandez M E Lopez-Gonzalez B Colaco and V Villar-Suarez ldquoStudy on viability and chondrogenic differentiationof cryopreserved adipose tissue-derived mesenchymal stromalcells for future use in regenerative medicinerdquo Cryobiology vol71 no 2 pp 256ndash263 2015

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


can be isolated easily from adipose tissue the cells areobtained through plastic surgery that represents an ampleand accessible source of adult stem cells with the ability todifferentiate into adipocytes osteocytes chondrocytes andcells phenotypically similar to neurons [5] Due to ADMSCability to differentiate into several cell types of clinical inter-est they hold an immense potential for the future therapeuticuse of these cells in clinics Although many reports relatedto the therapeutic potential of ADMSC most of the currentinvestigationswere performedmainly on freshly isolated cells[6] As this cell type holds a clinical translation value there isan increasing interest in stem cell banking of ADMSC

In consequence of the inherent characteristics of theADMSC low immunogenicity higher cellular yield incomparison to bone marrow cells and pluripotency thecryopreservation of these cells would be a very convenientalternative However the effects of the cryopreservationand the thawing proceedings could impact their therapeuticoutcome and therefore should be evaluated Once the cellsare cultivated and are attached to the plastic before thecells cryopreservation these are subjected to a subtractdesegregation stress being able to put in risk the integrityof the cell membrane and its surface molecules for the celladhesion After thawing the cryopreserved cells have also therisk of injuring the membrane and other cellular functionalcharacteristics both proceedings could affect the therapeuticoutcomes

In this context the present study aims to address whetherthe ADMSC cryopreservation could compromise the cellintegrity the expression of adhesion molecules andor thedifferentiation potential of ADMSC and other stem cellslineages

2 Material and Methods

21 Experimental Design Adipose-derived mesenchymalstem cells were obtained from adipose tissues of 12 adulthealthy donors (10 female and two male) by liposuctionperformed by a plastic surgeon with informed consentsignature from each patientThe cell isolation was performedby enzymatic digestion with collagenase type I Then thecells were cultured in DMENF12 supplemented with 10 ofcalf fetal serum 100 unitsmL of penicillin and 100 120583gmLof streptomycin Before and after cryopreservation thefollowing assays were performed colony forming unitsimmunophenotype and cellular viability with Annexin Vand 7-AAD analysis by flow cytometer as well as ADMSCdifferentiation to adipocytes and osteoblasts

22 Isolation of ADMSC The adipose tissue samples werecollected from a healthy donor after consent signature fromeach patient All the samples were collected from specializedplastic clinics using the liposuction technique After thesamples were collected they were cultured before 24 hoursusing the following protocol 50mL of each sample wasextensively washed with phosphate buffered saline (PBS)(Sigma St Louis MO USA) containing 300 unitsmL ofpenicillin and 300 120583gmL of streptomycin (Sigma St Louis

MO USA) (PBSPS) After washing the samples and debrisremoval these were placed in 50mL tubes with 0075 oftype I A collagenase (Sigma St Louis MO USA) preparedin PBSPS The cells were homogenized and incubated withagitation at 37∘C for 30 minutes After the incubation thetype I collagenase activity was ceased by adding the samevolume of Eagle modified from DulbeccoF12 (DMEMF12)(LGC Biotechnology Brazil) containing 10 of fetal calfserum (FCS) (GIBCO B Life Technologies Inc RockvilleUSA RL) as determined to be standard culture medium(SCM) Then the samples were centrifuged at 400 g for 10minutes The supernatant was discarded and the pellet wasresuspended in 10mLof PBSPS then another centrifugationwas made at 400 g for 10 minutes The supernatant wasdiscarded and the pellet was suspended in 5mL of culturemedium The cell suspension was filtered in cell strainer of100 120583M(BectonDickinsonUSA)The cell countwasmade bya hemocytometer and the utilization of Trypan Blue (SigmaSt Louis MO USA) (for the dilution of 1 1 10 120583L of the cellsuspension was added for 10 120583L of Trypan Blue) After that1 times 105 cells for cm2 in 25 cm2 and 75 cm2 culture flasks wereharvested and incubated at 37∘C and 5 of CO

2[7ndash9]

23 Cultivation and Expansion 72 hours after on cultivationthe medium was aspirated from the flasks and the cellswere washed with preheated PBSPS then 7mL of culturemedium was added in the 25 cm2 and 12mL in the 75 cm2flasks The cells were kept incubated at 37∘C and 5 CO

2

Themediumwas changed twice a week until the cells reachedbetween 80 and 90 of confluence After the desiredconfluence was reached the cells were briefly washed withpreheated PBSPS and added trypsinEDTA (025) (SigmaSt Louis MO USA) for disaggregation of the adherent cellsThe cells were incubated for 10 minutes at 37∘C Then thesame volume of SCM was added for the neutralization of thetrypsin action The cell suspension was transferred to sterilecentrifuge tube and centrifuged at 400 g for 10 minutes Thesupernatant was discarded and the cells were resuspended inapproximately 5mL of SCMThe cell count was realized witha hemocytometer chamber using Trypan Blue (1 1 dilution)[9] After the counting cells were once again harvested in75 cm2 flasks at a concentration of 1 times 103 cellscm2 andincubated at 37∘C and 5 of CO

2until they reached 80 of

confluence When they reached the desired confluence thecells were trypsinized andwere available for analysis For eachsample the culture was done at passage 2 (P2)

24 Cryopreservation Proceedings After trypsinization at P2cells as described previously and confirming their viabilityby the Trypan Blue exclusion method a fraction of the cellswere submitted to the cryopreservation proceedings Thecells were counted with a hemocytometer (approximately1 times 106 cellsmL) The cryoprotectant medium had 80 ofBFS 10 of dimethyl sulfoxide (DMSO) (Sigma St LouisMO USA) 10 of DMEM-F12 (LGC Biotechnology Brazil)and 100 unitsmLof penicillin and 100120583gmLof streptomycin(Sigma St Louis MO USA) 1mL aliquots (containing 1 times106 cells) were transferred to preidentified cryogenics tubes






















119901 lt 005











lowastlowast

50

60

70

80

90

100

110

Expr

essio

n (

)


3 Results






CD34 CD34 CD73 CD73

CD45 CD45 CD90 CD90



Before After

Annexin V Annexin V

7-AAD 7-AAD






4 Discussion




(a) (b)


(a) (b)

















5 Conclusions




Acknowledgment


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















119901 lt 005











lowastlowast

50

60

70

80

90

100

110

Expr

essio

n (

)


3 Results






CD34 CD34 CD73 CD73

CD45 CD45 CD90 CD90



Before After

Annexin V Annexin V

7-AAD 7-AAD






4 Discussion




(a) (b)


(a) (b)

















5 Conclusions




Acknowledgment


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



CD34 CD34 CD73 CD73

CD45 CD45 CD90 CD90



Before After

Annexin V Annexin V

7-AAD 7-AAD






4 Discussion




(a) (b)


(a) (b)

















5 Conclusions




Acknowledgment


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)


(a) (b)

















5 Conclusions




Acknowledgment


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














5 Conclusions




Acknowledgment


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Human Adipose-Derived...

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