pharmacol sci 2013;17:820-833 a novel dna profiling ...lized to repair or regenerate injured tissues...

Abstract. – BACKGROUND: Autologous chon-drocyte implantation (ACI) is a cell-based treat-ment that can be used to regenerate chondral de-fects. European legislation specifically classifiessuch produced chondrocytes as “medicinal foradvanced cell therapy” that have to be manufac-tured in pharmaceutical factories according tospecific rules, named Good Manufacturing Prac-tices (GMPs). One main requirement of cell manip-ulation in advanced therapy is to prevent the riskof any contamination.

AIM: The aim of this study was to verify ifchondrocyte cultures suitable for ACI were freeof cross-contamination by means of DNA profil-ing techniques.

MATERIALS AND METHODS: Cell cultureswere carried on in a Hospital Cell Factory incompliance with European current Good Manu-facturing Practices. DNA profiling, by means ofShort Tandem Repeats and miniShort TandemRepeats analyses, was performed on expandedchondrocytes and their related control bloodsamples. Mitochondrial DNA was analysed tofurther confirm the results and to evaluate pos-sible mutations occurred in the samples.

RESULTS: Our findings demonstrated the ab-sence of cross-contamination between chondro-cyte cultures and, thus, their identity mainte-nance until the end of the manipulation.

CONCLUSIONS: DNA profiling technique canbe a suitable test for quality control not only forchondrocyte manipulation, but for cell therapyin general.

Key Words:Advanced cell therapy, Autologous chondrocyte

implantation, Good Manufacturing Practices, Cross-contamination, DNA profiling.

Abbreviations

ACI = Autologous Chondrocyte ImplantationAIFA = Agenzia Italiana del Farmaco

European Review for Medical and Pharmacological Sciences

A novel DNA profiling application forthe monitoring of cross-contamination inautologous chondrocyte implantation

L. ROSETI, A. BASSI, P.M. FORNASARI1, M. SERRA, F. CANELLA,A. MASO, D. DALLARI2, C. BINI3, S. PELOTTI3

Cell Factory, and 1Musculoskeletal Cell and Tissue Bank; Prometeo, Rizzoli RIT (Research InnovationTechnology), Istituto Ortopedico Rizzoli, Bologna, Italy. 2Conservative Orthopaedic Surgery and In-novative Technique Ward, Istituto Ortopedico Rizzoli, Bologna, Italy3Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Italy

Corresponding Author: Livia Roseti, Ph.D; e-mail: [email protected]

bp = base pairBSE/TSE = Bovine Spongiform Encephalopathy/

Transmissible Spongiform EncephalopathyDMEM = Dulbecco’s Modified Eagle MediumDNA = Deoxyribonucleic acidEDTA = Ethylenediaminetetraacetic acidEU-cGMPs = European current GMPsEU/ml = Endotoxin Units per mlFBS = Foetal bovine serumFDA = Food and Drug AdministrationGMP = Good Manufacturing PracticesHIV = Human Immunodeficiency VirusHCV = Hepatitis C VirusHBV = Hepatitis B VirusLAL = Lymulus Amebocyte LysatePCR = Polymerase Chain ReactionPES = probabilistic expert systemsQC = Quality controlRMP = Random Match ProbabilityrRNA = ribosomal Ribonucleic AcidrRCS = revised reference sequenceSTR = Short Tandem Repeat

Introduction

Cell-based therapies have existed since thefirst successful bone marrow transplantation in19681,2. The subsequent increased understandingof cell biology at protein, molecular and geneticlevels and the development of techniques such astissue engineering and concurrent studies in thefield of scaffold design have expanded the hori-zon of likely therapeutic uses3. Cell-based thera-pies have evolved during last years and are nowapplied in an increasing numbers of fields and inseveral clinical trials for congenital and acquireddisorders. Public expectation for such noveltreatments is high, but there have been only fewcompleted trials. Moreover, their full potentialstill remains to be clarified and aspects like long-

2013; 17: 820-833

820

term outcomes or tumorigenesis/cancerogenesisneed to be more in depth investigated and com-pleted. This complex situation including differentstakeholders (academia, clinicians, patients, pub-lic structures, enterprises) has raised the need todevelop regulatory frameworks to guarantee pa-tient safety and efficacy4.

In the musculoskeletal system, there is a widevariety of cell-based applications5. Cells are uti-lized to repair or regenerate injured tissues (carti-lage, bones, tendons, ligaments, muscles, etc.), orto treat chronic conditions such as rheumatoidarthritis. From recent studies, it appears that, inEurope, in general, autologous cells are predomi-nantly used6,7. Newly, advances in gene deliverytechnology have created the additional opportu-nity to treat genetic diseases like Duchenne mus-cular dystrophy (DMD) with gene therapy8.

To date, proposed employments for cell-basedmedicinal products in regenerative medicine arequite impressive9. Autologous chondrocyte im-plantation (ACI) is a cell-based treatment thatcan be used as second-line measure to regeneratechondral or osteochondral defects in younger, ac-tive patients10. It is a widely diffused techniqueand many groups have reported good results bothfrom the histological and clinical point of view.Nevertheless, there is still scepticism about ACIclinical and cost effectiveness, especially in com-parison with other traditional treatments. Litera-ture revisions highlights the need of further trialswith long-term follow up11,12.

It is known that hyaline articular cartilage in-juries may lead to pain and loss of function due totissue’s limited capacity for self-repair13. Such le-sions predispose individuals to osteoarthritis inlater life and eventually to requirements for totaljoint replacement. This is in general associatedwith a significant impact on quality of life andrepresents a huge socioeconomic burden to soci-ety. ACI approach was firstly introduced by Brit-tberg et al14 to treat full-thickness chondral de-fects of the knee. The treatment was later appliedto the ankle15 and it is now suitable also for otherjoints such as hip16. In the original proceduresmall grafts of normal cartilage removed fromnon weight bearing areas of the knee were treatedin a proper laboratory to obtain chondrocytes.The cells were expanded in monolayer in suitablemedia and the suspension injected into the pre-pared defects a few weeks later. Clinical, radio-logical and histological results are available at 10to 20 years after the implantation, suggesting thatoutcomes remain high, with relatively few com-

plications17. Recent generation technique includesthe additional step to culture chondrocytes ontoscaffolds which act as carriers ensuring spatialcell distribution and phenotype stability. The en-gineered tissues are then cut to the correct sizeand shape of the defects. The scaffolds, which ef-ficiently “mimic” the natural surroundings of car-tilage cells, may have different origins (synthetic,natural) and characteristics (bi/three dimensionalstructures, gels, sponges, microspheres, etc.)18.

It appears that in vitro chondrocyte manipula-tion is a crucial phase of ACI as long as the sur-gical one. Current European legislation specifi-cally defines such manipulation as “extensive”and classifies such produced chondrocytes as“medicinal for advanced cell therapy” (Regula-tion (EC) No 1394/2007 of the European Parlia-ment and of the Council on advanced therapymedicinal products and amending Directive2001/83/EC and Regulation (EC) No 726/2004).Those are not only definitions, but imply specifictechnical and practical consequences. In fact,such cell-based products have to be manufac-tured in pharmaceutical factories and accordingto specific rules, named Good ManufacturingPractices (GMPs) (European Commission, TheRules Governing Medicinal Products in the Eu-ropean Union. Volume 4-Guidelines for goodmanufacturing practices for medicinal productsfor human and veterinary use. Current Edition)that are currently utilized for the pharmaceuticalproducts. Moreover, in the European Union,manufacturing shall be authorised by the compe-tent authority of each Member State.

Inside Rizzoli Orthopaedic Institute, Bologna,Italy, is located a “Cell Factory” authorized bythe Italian Drug Agency (Agenzia Italiana delFarmaco, AIFA) since 2009, working accord-ing to current GMPs (cGMPs) and manipulat-ing chondrocytes for clinical use (ACI). Toreach cGMPs compliance and gain the Autho-rization we carefully designed and validated achondrocyte manufacturing process ensuringproducts consistency (microbial and viral in-tegrity, viability purity, identity, yield and sta-bility) (Reflection paper on in-vitro culturedchondrocyte containing products for cartilagerepair of the knee, London, 08 April2010EMA/CAT/CPWP/568181/2009, Commit-tee For Advanced Therapies). Finally, as specifi-cally requested by GMPs, we had also to demon-strate that all the measures adopted to prevent thepossibility of cross-contamination of each pro-cessing medicinal lot were effective.

821

DNA profiling in autologous chondrocyte implantation

822

L. Roseti, A. Bassi, P.M. Fornasari, M. Serra, F. Canella, A. Maso, D. Dallari, C. Bini, S. Pelotti

Figure 1. Production facility (Cell Factory) of Rizzoli Orthopaedic Institute. A, Pass box to introduce raw materials into theclean room. B, Biohazard Hood (Grade A area) for high risk operations like cell manipulations. C, and D, Grade B workplaces for lower risk operations such as media warming and centrifugation. Air classification is monitored in real time by ameasuring system as indicated by the arrows.

In this paper we describe the study aimed toverify if chondrocyte cultures produced in ourCell Factory and suitable for autologous implan-tation were free of cross-contamination by meansof a novel application of DNA profiling utilizedin human identification for forensic purpose.

Materials and Methods

Manipulation Areas: Clean RoomsFor this study, chondrocyte manipulations

were performed in a production facility (Cell Fac-tory) located inside our Institute (Figure 1). Thestructure includes two clean rooms of differentclassification up to A (the local zone for high riskoperations i.e. Biohazard Hood) in B work places(the background environment for the grade Azone), according to European current GMPs (EU-cGMPs). To minimize traffic and contaminationentry into the clean room, raw materials are intro-duced separately from personnel through a cleanpass box (Figure 1A). High risk operations in-cludes manipulations where the cells are exposed

to environment such as trypsinization, mediumchange, biomaterial seeding (Figure 1B). Forlower risk operations, such as media warming orcentrifugation, grade B areas are sufficient (Fig-ure 1C and D). Only trained and equipped per-sonnel is admitted in the structure. Operator’sequipment includes protective and disposableclothes to wear. As required by EU-cGMPs, inthe Cell Factory various factors, including air-borne contaminants temperature, relative humidi-ty and differential pressure and static electricityare kept under strict control by a real time mea-suring system (A&LCO Industries, ColognoMonzese, Milano, Italy; Pharmaceuticals Net 3.2facility Monitoring Software, Boulder, CO, USA)(arrows in Figures 1B and C).

ReagentsReagents choice was geared towards products

suitable for cell therapy applications, ensuringhigh quality performances. In particular, we uti-lized a foetal bovine serum (FBS) that was certi-fied to be produced in Australia and free frombovine spongiform encephalopathy/transmissible

spongiform encephalopathy (BSE/TSE) (Euro-pean Agency for the Evaluation of Medicinalproducts/Committee for Proprietary medicinalproducts. Note for Guidance on Use of BovineSerum in the Manufacture of Human BiologicalMedicinal Product (CPMP/BWP/1793/02), 2002).

All reagents were all cross-checked by internalquality controls in the Cell Factory and, only af-ter passing such tests, they were considered to beadequate for the manipulation process.

Donor ScreeningWe utilized cells from eight patients (here

named Patient 1, Patient 2, Patient 3, Patient 4, Pa-tient 5, Patient 6, Patient 7 and Patient 8) undergo-ing ACI. Informed consent was obtained from allpatients who entered the study and the ACI proce-dure was approved by the Ethical Committee ofIstituto Ortopedico Rizzoli, Bologna, Italy. Bloodsamples harvested from the patients duringarthroscopy were used to evaluate the presence oftransmissible pathologies. The same minimum setof testing requirements was applied as for allo-geneic living donors: HIV-1/HIV-2 Antibody, He-patitis B Surface Antigen, Hepatitis B Core Anti-body, Hepatitic C Virus Antibody and test forsyphilis. In addiction, Nucleic Acid Techniques(NAT) for HBV DNA, HCV RNA, HIV DNA de-tection were performed to reveal virus presencealso during the window period (Directive2004/23/EC Directive 2004/23/EC of the Euro-pean Parliament and of the Council of 31 March2004 on setting standards of quality and safety forthe donation, procurement, testing, processing,preservation, storage and distribution of humantissues and cells). Since our structure does nothave the possibilty to manipulate the infected cellsin separated areas positive patients must be ex-cluded from our study.

Chondrocyte cGMPs Manipulation andBatch Release

Cartilage biopsies, removed arthroscopicallyfrom non-weight-bearing areas on the femoralcondyles in the operating room, were introducedin the Cell Factory clean rooms for manipulation.After weighing, samples were minced with ascalpel and carefully washed with cell culturemedium Dulbecco’s Modified Eagle Medium(DMEM ) high glucose (4.5 g/L) GMP Grade (LiStarFish, Carugate, Milan, Italy), supplementedwith 10% FBS GMP Grade (Li StarFish ) and L-Glutamine 4 mM (complete medium) (LiStarFish). No growth factors, cytokines or other

supplements were added. The chondrocytes werethen isolated by enzymatic digestion withTrypsin-EDTA (1:250) GMP Grade (Li StarFish)15 minutes and 740 U/mL Collagenase II (LiStarFish) at 37°C, 5% CO2, 95% RH, for 22hours. Complete medium was added and, aftercentrifugation, cells were seeded at low density(BD Falcon™ vented cell culture flasks). Primarycultures were expanded in monolayer up to pas-sage 3: medium was changed twice weekly and atconfluence cells were trypsinized in larger flasks.

Cell morphology was monitored during expan-sion with inverted contrast phase microscope(Eclipse TE200, Nikon Instrument S.P.A., Flo-rence, Italy) and pictures were taken at passage 0and 3 with a DS-Fi1 digital camera.

Chondrocytes were then seeded onto a bioma-terial derived from porcine collagen I/III, namedChondro-Gide (Geistlich Biomateriale, Woll-husen, Switzerland)19, at a density of 0.5x106

cells/cm2. This seeding low limit has been estab-lished by previous studies performed in our Lab-oratory. To assess cell number and viability aNucleoCounter Automatic Cell Counting Sys-tem (Sartorius Stedim, Biotech, Goettingen,Germany) was used. The system is able to detectsignals from the fluorescent dye propidium io-dide (PI) bound to cell nuclei. Cell viability ac-ceptance limit was: ≥ 80%. After 3-5 days inculture inside the biomaterial the engineered tis-sues were double packaged and released.

Quality Control (QC)QC analyses, complying with cGMPs and Eu-

ropean Pharmacopeia (European Pharmacopoeia,Current Edition), were performed to assess themicrobial load of the biopsy following collec-tion, throughout the chondrocyte manipulationprocess and at the time of release for clinical use.

Sterility TestingThe technology used was BacT/Alert 3D, Auto-

mated Microbial Detection System (BiomerieuxIndustry, Marcy L’Etoile, Craponne, France) thatallows to reveal the presence of aerobic, anaerobicbacteria and fungi. The System has been alreadyvalidated by FDA for chondrocyte culture moni-toring in autologous implantation20. At each test-ing point, 1 ml of cell culture surnatants was inoc-ulated in bottles containing specific bacterialgrowth media. The bottles were then incubated inthe BacT/alert System for 7 days.. If there is a mi-crobial growth inside the bottles, CO2 will be pro-duced (due to substrate metabolization) inducing a

823


824

colorimetric change in the culture medium (due toa change in pH medium) that is registered by theDetection System. If there is no microbial growthand thus no colorimetric change, the sample isconsidered negative.

Mycoplasma TestingAnalyses for Mycoplasma contamination detec-

tion were performed both on surnatants (0.5 ml)and on chondrocytes (50.000 cells) to even evaluateits presence inside the cells. Highly-sensitive RealTime Quantitative DNA PCR technology was themethod used for detecting contaminating My-coplasma. DNA extractions were performed with“High Pure PCR Template Preparation kit” (Roche,Applied Science, Basel, Switzerland), amplifica-tions with Venor®GeM-qDual Mycoplasma Detec-tion Kit for Real-Time PCR (Minerva BiolabsGmbH, Berlin, Germany). This Kit displays a de-tection range of more than 25 Mycoplasma speciessince primers are specific for a common region ofthe 16S rRNA gene. To detect amplicon a specificfluorescent SCORPION probe was utilized (Miner-va Biolabs GmbH, Berlin, Germany). Real TimePCR reactions were carried out in a LightCycler®

2.0 instruments (Roche, Applied Science, Basel,Switzerland). All samples were tested in parallelwith positive and negative controls, as well as withan internal amplification control.

Endotoxin TestingBacterial endotoxin assays were performed on

final product surnatants (0.5 ml) by quantitativeLymulus Amebocyte Lysate (LAL), test using theEndosafe®-PTS (Charles River Laboratoires,l’Arbresle, France). This System has been li-censed by the FDA as a method for release test-ing of pharmaceuticals products. Test acceptancelimit was < 0.5 Endotoxin Units per ml (EU/ml).

DNA Profiling Analysis

SamplesAn aliquot of blood sample harvested from

each patient during arthroscopy and a pelletedaliquot of chondrocytes (80.000 cells) from eachculture before the seeding onto the biomaterialwere frozen for genetic testing.

DNA ProfilingDNA was extracted by QIAmp Mini Kit follow-

ing manufacturer’s instructions (Qiagen, Hilder,Germany). Extracted DNA was visualized byelectrophoresis on 1% agarose gel containing

ethidium bromide. 4 ng of DNA template weresubmitted to fluorescent multiplex polymerasechain reaction (PCR) to amplify 15 tetranucleotideShort Tandem Repeat (STR) loci (molecular size:107-358bp): D8S1179, D21S11, D7S820,CSF1PO, D3S1358, THO1, D13S357, D16S539,D2S1338, D19S433, vWA, TPOX, D18S51,D5S818, FGA and amelogenin included in theAmpFlSTR Identifiler PCR Amplification Kit(Applied Biosystems, Life Technologies, Carls-bad, CA, USA)21 according to the manufacturer’srecommendations. Amelogenin locus for sex de-termination was also useful as internal control.

Nine miniSTRs of AmpFlSTR MiniFiler PCRAmplification Kit (Applied Biosystems), whoseamplicons range from 70 to 283 nucleotides, wereanalyzed with 2 ng of DNA template, according tothe manufacturer’s recommendations22: D13S317,D7S820, D21S11, D2S1338, D16S539, D18S51,CSF1PO, FGA and amelogenin.

Mitochondrial DNA (mtDNA) analysis wasperformed in a reaction volume of 25 ml contain-ing: 10 ng of genomic DNA, 1X PCR buffer, 1.5µM MgCl2, 200 mM of each dNTP, 1.5 U Am-pliTaq DNA Polymerase (Applied Biosystems),0.2 mM of each primer (L15997-H16401, L29-H408). The amplification was carried out for 35cycles: 3 min at 94°C, 1 min at 94°C, 30 sec at56°C and 1 min at 72°C, with a final extension of10 min at 72°C. PCR products were run on a 2%agarose gel and stained with ethidium bromide.Amplicons were purified by ExoSAP-ITTMreagent (USB Corporation, Cleveland, OH,USA) according to the manufacturer’s protocol.Sequencing was performed using BigDye Termi-nator Cycle Sequencing Kit v 1.1 (AppliedBiosystems, USA) and sequence products werepurified by ethanolic precipitation.

Dye-labeled PCR fragments were analyzed bycapillary electrophoresis using an ABI PRISM310 Genetic Analyzer (Applied Biosystems) andsequences were aligned and compared with therevised reference sequence (rRCS) using the Se-quence Navigator computer program (AppliedBiosystems, Sequence Navigator version 1.0.1).

Statistical AnalysisThe calculation of Random Match Probability

(RMP) based on genotype frequencies23 was esti-mated using the Italian database24.

Mixed SamplesThree cultures were carried out in our Cell

Factory in the same period of time. One chon-


825


Figure 2. Morphological changes of monolayer expanded chondrocytes. Picture of a representative patient’s chondrocytesmonolayer. Morphological observations revealed that, in general, chondrocytes were behaving similarly. A, At passage 0 thecells resembled polygonal while appeared in a more spindle morphology at passage 3 (B). Inverted contrast phase microscopeimages were taken with a digital camera (magnification × 20).

A B

Results

Donor ScreeningNone of the ten patients’ blood samples har-

vested at the time of biopsy arthroscopy revealedthe presence of transmissible pathologies. All thebiopsies were allowed to enter the Cell Factoryand thus manipulated.

Chondrocyte cGMPs ManipulationBiopsies weight ranged from 238 to 352 mg.Morphological observations of each patient’s

monolayer cultures revealed that, in general,chondrocytes were behaving similarly: at pas-sage 0 the cells resemble more polygonal (Figure2 A), while appeared in a more spindle/elongatedmorphology at passage 3 (Figure 2 B).

Chondrocyte viability ranged from 88% to99% at the time of release, with a mean of 92.5%and a Standard Deviation of 6.36%.

Quality ControlAll the cartilage biopsies were negative for

sterility testing and Mycoplasma detection.Sterility was maintained throughout all chondro-cyte manipulation processes and at the time ofrelease for clinical use. Endotoxin tests were <0.1 EU/ml in all cases.

DNA ProfilingThe eight samples analysed from autologous

chondrocytes cultures showed for STRs (Table Iand Figure 3) typing a unique DNA profilematching the reference sample without extrapeaks. The random match probability was esti-

drocyte culture was manipulated followingGMPs rules, as described above, and releasedfor clinical use (Patient 8). The others were twobone marrow Mesenchymal Stem Cell cultures(named Test 1 MSC and Test 2 MSC) utilizedonly for validation purposes and discarded atthe end of processing. Briefly, bone marrow as-pirates harvested from iliac crest with an opti-mized harvesting technique25 were derived fromtwo donors (median age 22) undergoing an or-thopaedic treatment. Informed consent allowedus to use an aliquot of each bone marrow aspi-rates for our study. Mononuclear cells were iso-lated and MSCs cultures characterized as de-scribed elsewhere26. Donor screening and MSCsmanipulations were performed in compliancewith current directives and GMPs, as describedabove.

Cell samples were harvested at the end of manip-ulation of the three cultures, scheduled the same day.At first we manipulated the chondrocyte culture (Pa-tient 8), harvesting the sample for DNA profiling.Then we manipulated Test 1 MSC culture harvest-ing two samples for DNA profiling one of whichwas maintained open (and named Test 1 MSC) un-der the laminar flow until the end of Test 2 MSCmanipulation, the other was closed. Finally, we ma-nipulated Test 2 MSC culture harvesting again twosamples for DNA profiling, one of which was closed(and named Test 2 MSC), the other was mixed(same number of cells) with the closed sample of theprevious culture and named Test 1 & Test 2 MSC.

Cell samples and their related blood sampleswere pelleted and analyzed for STRs, miniSTRsand mtDNA as described above.

826


Figure 3. Electropherograms of PCR products in a representative patient (Patient 5). Eight engineered chondrocyte and relat-ed patient blood samples analyzed by means of DNA profiling in order to evaluate cross-contamination during GMPs manipu-lation. The picture of representative Patient 5 shows that engineered chondrocytes DNA profile (A) is unique matching theblood reference sample profile (B) without the presence of extra peaks corresponding to alleles revealing human exogenousDNA from other biological sources.

Mixed SampleTest 1 and Test 2 MSC mixed samples showed

a profile compatible with both its components(Table III and Figure 4). In particular, STRsanalysis revealed a mixed genetic profile, enclos-ing both Test 1 and Test 2 MSC allelic compo-nents at 50% each. Applying a biostatistic calcula-tion by means of the DNA mixture method (Fungand Hu, J. R. Statistic Society, 2000) we found alikelihood value of 6.5 × 1015. This value stronglysupports the hypothesis of a mixture of Test 1 and

mated in a range from 1×10-15 to 1×10-18. Resultswere confirmed also by miniSTRs typing (datanot shown).

Chondrocyte sequence of mitochondrialDNA HVI and HVII control regions showed anidentical haplotype without heteroplasmic nu-cleotide positions neither de novo mutationscompared to blood reference samples se-quences and revealed the variations indicatedin Table II, with respect to Anderson referenceSequence.

827


Patien

t1

Patien

t2

Patien

t3

Patien

t4

Patien

t5

Patien

t6

Patien

t7

Patien

t8

Auto

som

icm

arke

rs(c

ells

)(b

lood)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

(cel

ls)

(blo

od)

D8S

1179

10-1

310

-13

13-1

413

-14

13-1

413

-14

11-1

111

-11

10-1

010

-10

14-1

414

-14

10-1

410

-14

14-1

514

-15

D21

S11

30-3

1.2

30-3

1.2

28-3

028

-30

28-2

928

-29

28-2

928

-29

30.2

-32.

230

.2-3

2.2

28-2

828

-28

28-3

028

-30

28-3

128

-31

D7S

820

11-1

311

-13

10-1

210

-12

11-1

111

-11

11-1

111

-11

9-11

9-11

10-1

010

-10

8-12

8-12

8-8

8-8

CSF

1PO

10-1

010

-10

10-1

010

-10

11-1

311

-13

11-1

211

-12

11-1

311

-13

7-10

7-10

9-11

9-11

10-1

210

-12

D3S

1358

15-1

815

-18

15-1

515

-15

15-1

815

-18

15-1

615

-16

16-1

716

-17

15-1

715

-17

16-1

816

-18

15-1

715

-17

TH01

6-9

6-9

8-9

8-9

6-8

6-8

7-7

7-7

7-9

7-9

7-7

7-7

8-9.

38-

9.3

6-9.

36-

9.3

D13

S317

11-1

311

-13

8-13

8-13

11-1

211

-12

11-1

111

-11

11-1

311

-13

11-1

211

-12

11-1

211

-12

8-11

8-11

D16

S539

11-1

311

-13

11-1

111

-11

12-1

212

-12

12-1

212

-12

9-13

9-13

9-11

9-11

10-1

010

-10

11-1

211

-12

D2S

1338

17-2

317

-23

17-1

717

-17

24-2

524

-25

24-2

524

-25

19-2

519

-25

17-1

817

-18

20-2

520

-25

19-2

319

-23

D19

S433

14-1

514

-15

15-1

5.2

15-1

5.2

13-1

413

-14

13-1

413

-14

14-1

514

-15

12.2

-14

12.2

-14

14-1

514

-15

13-1

413

-14

vWA

17-1

917

-19

16-1

716

-17

16-1

716

-17

17-1

917

-19

18-1

818

-18

14-1

614

-16

17-1

717

-17

17-1

817

-18

TPO

X8-

88-

88-

88-

88-

118-

119-

109-

108-

98-

98-

98-

99-

119-

118-

98-

9D

18S5

112

-18

12-1

813

-16

13-1

614

-16

14-1

618

-19

18-1

914

-18

14-1

814

-16

14-1

613

-14

13-1

413

-17

13-1

7A

MEL

XY

XY

XY

XY

XX

XX

XY

XY

XY

XY

XY

XY

XY

XY

XY

XY

D5S

818

11-1

211

-12

11-1

211

-12

9-12

9-12

12-1

212

-12

11-1

311

-13

11-1

311

-13

10-1

110

-11

13-1

313

-13

FGA

21-2

221

-22

21-2

221

-22

20-2

220

-22

22-2

622

-26

25-2

625

-26

23-2

523

-25

20-2

320

-23

20-2

020

-20

Table

I.D

NA

prof

iling

anal

ysis

byST

Rs

onG

MPs

man

ipul

ated

chon

droc

ytes

(cel

ls)

and

rela

ted

cont

rols

ampl

es(b

lood

).

828

Test 2 samples rather that of two individuals takenat random in the population. Mitochondrial DNAsequencing revealed point heteroplasmies at thetwo hyper variable regions HV1 and HV2.

Patient 8 (chondrocytes), Test 1 MSC (the oneleft open during Test 2 MSC manipulation) andTest 2 MSC showed unique DNA profile both atnuclear and mitochondrial level (Table III A andB, respectively), in line with the results alreadyobtained with the chondrocyte cultures.

Discussion

The term cross-contamination is used to indi-cate misidentification of one cell line by another,rather than contamination by microbiological or-ganisms27. Recently, a role for gene profilinganalysis in the authentication of human cell lineshas been proposed28,29. Despite the still scarce lit-erature, this is a serious and often unrecognizedproblem since it can compromise the reliability ofscientific findings30,31. Such studies are mostly inthe research field, concerning contamination com-ing from tumor cell cultures, while the possibilityof mix-up between normal cells (from other typesof cultures or from the same type but different pa-tients) usually has little or no consideration. Therisk of cross-contamination between cultures andthe consequent loss of identity exists not only inthe research field, but also for cell therapy, such asACI, where it is obviously more relevant.

GMPs have been established by legislator pre-cisely to guarantee the quality of pharmaceuticalsproducts, including cell-based ones. The first stepto this goal is the environment that must be builtfollowing specific and mandatory fees. A clean-room is a place where different parameters such asair filtration and ventilation conditions, tempera-ture, relative humidity, differential pressure, num-ber of air particles and Colony Forming Units arestandardized and constantly monitored. Produc-tion operations must follow clearly defined proce-dures. Standardized processes, analytical methodvalidations, defined cleaning procedure, personnelflow and training and process traceability shouldbe developed and performed. High qualityreagents and plastic ware, disposable materials,“closed systems” should be used. At every stageof processing, cells should be protected from mi-crobial or other contaminants. Different cell-basedproducts should not be manipulated simultaneous-ly or consecutively in the same room, unless thereis no risk of mix-up or cross-contamination.

Our facility is a public structure that was au-thorized for GMP Production for Advanced CellTherapy by the Italian Drug Agency (AgenziaItaliana del Farmaco, A.I.F.A.) in 2009. Weadopt all the appropriate and mandatory organi-zational and technical measures foreseen by theGMP to prevent cross-contamination betweenbatches. However, our situation can be consid-ered more subject to this risk in comparison withbig enterprise’s structure: small dimensionsmake difficult to have segregated areas and fullymanual productions does not permit the use of“closed systems” (it has to be mentioned that theuse of automated production lines does not solveevery problem: instruments are considered a ma-jor source of cross-contamination during produc-tion). Even if we adopted other measures like theuse of disposable materials and lots productionby campaign (separation in time) followed by ap-propriate and validated cleaning, the Italian au-thority claimed an evidence that cross-contami-nation was not present in the manipulated chon-drocytes cultures. We decided to apply the DNAprofiling technique, a high sensitive and specificmethod already used in forensic caseworks forhuman identification32 and also applied inhematopoietic stem cell transplantation to revealand monitor microchimerism33.

DNA profiling is the determination in an indi-vidual of unique genetic characteristics that makeit distinguishable from of all other humans, in-cluding closely related ones. Identification is per-formed using validated commercial kits for spe-cific molecular markers such as microsatellite,also known as STR loci, which represent hyper-variable regions of DNA composed of tandemrepeats of 4 bp core nucleotide sequences. TheseDNA markers display only a specificity for hu-mans with no amplification of contaminant DNAsuch as fungal and bacterial sources34. Further-more, in forensic field very low amounts of DNAcan be more successfully typed with redesignedprimers to obtain reduced sized PCR products(miniSTRs)35. Mitochondrial DNA, being a lin-eage marker since only maternally inherited,doesn’t allow individual identification, but it canbe used to further confirm DNA profiling results.

The statistical approach for the cell source at-tribution to the patient is based on the randommatch probability calculation equated with theprobability that a match would occur by chance.This random match probability is the unlikelycoincidence that an unrelated person would bychance have the same DNA profile and can be


829


Hyp

erva

riab

lere

gio

ns

Pat

ient1

Pat

ient2

Pat

ient3

Pat

ient4

Pat

ient5

Pat

ient6

Pat

ient7

Patien

t8

HV

116

234T

,16

069T

,16

169T

,16

069T

,16

126C

,16

126C

,A

nder

son

1630

4C16

270T

1612

6C,

1618

3C,

1612

6C16

163G

,16

187T

,16

193T

,16

189C

,16

186T

,16

189C

,16

217C

1622

3T,

1618

9C,

1622

3T,

1627

8T,

1629

4T16

264T

,16

297C

1627

0T,

1627

6T,

1631

1CH

V2

263G

,73

G,

73G

,73

G,

73G

,73

G,

263G

,26

3G,

309.

1C,

150T

,15

3G,

185A

,26

3G,

152C

,31

5.1i

nsC

315.

1C31

5.1C

152C

,19

5C,

228A

,30

9.1i

nsC

,18

2T,

263G

,22

5A,

263G

,31

5.1i

nsC

185T

,29

5T,

226C

,29

5T,

195C

,31

5.1C

263G

,31

5.1i

nsC

198T

,30

9.1C

,24

7A,

315.

1C26

3G,3

15.1

insC

,35

7G

Table

II.M

itoch

ondr

ialD

NA

HV

Ian

dH

VII

cont

rolr

egio

nsan

alys

esin

both

chon

droc

ytes

and

bloo

dsa

mpl

es.

HV

:Hyp

erva

riab

leR

egio

n.

830


A. Nuclear DNA

Test 1 MSC Test 2 MSC Test 1 and test 2 MSC Patient 8Autosomicmarkers (cells) (blood) (cells) (blood) (cells) (cells) (blood)

D8S1179 10-13 10-13 14-14 14-14 10-13-14 14-15 14-15D21S11 30-34.2 30-34.2 29-33.2 29-33.2 29-30-33.2-34.2 28-31 28-31D7S820 10-11 10-11 9-12 9-12 9-10-11-12 8-8 8-8CSF1PO 10-12 10-12 10-13 10-13 10-12-13 10-12 10-12D3S1358 15-17 15-17 15-17 15-17 15-17 15-17 15-17TH01 6-9 6-9 7-7 7-7 6-7-9 6-9.3 6-9.3D13S317 12-12 12-12 11-11 11-11 11-12 8-11 8-11D16S539 12-12 12-12 12-14 12-14 12-14 11-12 11-12D2S1338 17-21 17-21 17-17 17-17 17-21 19-23 19-23D19S433 14-15 14-15 13-14 13-14 13-14-15 13-14 13-14vWA 16-16 16-16 15-16 15-16 15-16 17-18 17-18TPOX 8-11 8-11 8-10 8-10 8-10-11 8-9 8-9D18S51 13-18 13-18 14-16 14-16 13-14-16-18 13-17 13-17AMEL XX XX XY XY XY XY XYD5S818 11-13 11-13 11-12 11-12 11-12-13 13-13 13-13FGA 22-23.2 22-23.2 21-22.2 21-22.2 21-22-22.2-23.2 20-20 20-20

B. Mitochondrial DNA in both chondrocytes and blood samples

Hypervariableregion Test 1 MSC Test 2 MSC Test 1 and test 2 MSC Patient 8

HV1 16224C, 16192T, 16192C/T, 16304C16261T, 16256T, 16224C/T,16311C 16270T 16256C/T,

16261C/T,16270T/C,16311C/T

HV2 73G, 150T, 73G, 263G, 73G, 150T/C, 263G, 315.1C195C, 263G, 309.1C, 315.1C 195T/C, 263G,

309.1C, 315.1C 309.1C, 315.1C

determined by calculating the frequency of theobserved profile in a reference population data-base. The estimated values for a set of 15 STRsare generally beyond one in billions or trillions tonumbers that are not frequently used becausethey are so large36, values of the same magnitudefound in the present study.

Even if our results indicated the absence ofcross-contamination during manipulation in ourCell Factory, we decided to test the strength of oursystem, as in general required by GMPs. We veri-fied if the chondrocyte cultures were free of cross-contamination even when other cell types, such asMSCs, were present in the Cell Factory. For obvi-ous ethical reasons and to preserve at the mean-

time the chondrocyte culture we manipulatedMSCs in fully GMPs conditions and we waiteduntil results before chondrocyte release for clinicaluse. The results indicated that cross-contaminationwas present only in the samples created by mixingon purpose the two MSCs cultures. The sampleleft open during manipulation of the other MSCculture (Test 1 MSC) was negative, confirmingthe validity of our system. These results do notmean that GMPs can be escaped, but that ourprocess and our structure are robust enough toguarantee sterility and quality of the products, thuspatient’s safety. Moreover, they seem importantfor building a risk-based approach. In case of acci-dental contamination between batches there is no

Table III. DNA profiling of three non-mixed samples and the mixed sample.

MCS: Mesenchymal stem cell; HV: Hypervariable Region.

831


Figure 4. Electropherogram of PCR products in Test 1 & Test 2 mixed sample. STRs analysis performed on a mixed mes-enchymal stem cells culture sample (Test 1 & Test 2 MSC) showing a DNA profile compatible with both components.

Further studies are needed in order to explorethe potential of mtDNA mutations that seem tobe related with rapidly diving cells in tumors37.Mitochondrial DNA, being a lineage markersince only maternally inherited, doesn’t allow in-dividual identification, but it can be used to fur-ther confirm DNA profiling results.

This could be important especially for stemcell-based therapies that are now used to treatdifferent pathologies, but all the associated risksof in vitro or in vivo oncogenic transformationsare still not well known or understood, especiallyfor the long term follow-up38.

Conclusions

Since advanced cell therapy is becoming in-creasingly diffuse all over the world, DNA pro-filing could be a method of choice ideal for qual-ity control analysis in a Cell Factory working incGMPs compliance.

way to use them in patients, but they have to beeliminated. Special cleaning procedure should beapplied and an investigation performed in order tofind the causes, if any, and prevent or reduce thisrisk in the future.

This study suggests a novel application of DNAprofiling in ACI procedure since it allows to de-tect cross-contamination between cultures and toverify the maintenance of their identity until theend of the ex vivo process. The technique has sev-eral advantages respect to the older ones such aschromosome banding: it is faster, giving the possi-bility to obtain the results before implantation, it ismore precise allowing also the detection of smallamounts of contaminating DNA and it needs onlya few cells for the analysis.

Besides culture identification, our data showedalso that the genetic profile of each patient de-tected in blood sample controls before manipula-tion remains unaltered in the cells at the end ofthe process. Thus it seems that, at list in the ana-lyzed loci, our expansion process does not causegenomic alterations.

832

––––––––––––––––––––AcknowledgementsThis work was supported by the grant “Progetto di MedicinaRigenerativa” from “Regione Emilia Romagna” (Deliberadi Giunta n. 2233/2008).

References

1) BACH FH, ALBERTINI RJ, JOO P, ANDERSON JL, BORTIN

MM. Bone marrow transplantation in a patientwith the Wiskott-Aldrich syndrome. Lancet 1968;2: 1364-1366.

2) GATTI RA, MEUWISSEN HJ, ALLEN HD, HONG R, GOOD

RA. Immunological reconstitution of sex-linkedlymphopenic immunological deficiency. Lancet1968; 2: 1366-1369.

3) PARK DH, BORLONGAN CV, EVE DJ, SANBERG PR. Theemerging field of cell and tissue engineering. MedSci Monit 2008; 14: 206-220.

4) BELARDELLI F, RIZZA P, MORETTI F, CARELLA C, GALLI

MC, MIGLIACCIO G. Translational research on ad-vanced therapies. Ann Ist Super Sanità 2011;47: 72-78.

5) NÖTH U, RACKWITZ L, STEINERT AF, TUAN RS. Cell de-livery therapeutics for musculoskeletal regenera-tion. Adv Drug Deliv Rev 2010; 62: 765-783.

6) MARTIN I, BALDOMERO H, BOCELLI-TYNDALL C, SLAPER-CORTENBACH I, PASSWEG J, TYNDALL A. The survey oncellular and engineered tissue therapies in Eu-rope in 2009. Tissue Eng Part A 2011; 17: 2221-2230.

7) MASON C, DUNNILL P. Assessing the value of autol-ogous and allogeneic cells for regenerative medi-cine. Regen Med 2009; 4: 835-853.

8) DUAN D. Duchenne muscular dystrophy genetherapy: Lost in translation? Res Rep Biol 2011;2011: 31-42.

9) MASON C, MANZOTTI E. Regenerative medicine celltherapies: numbers of units manufactured andpatients treated between 1988 and 2010. RegenMed 2010; 5: 307-313.

10) HARRIS JD, SISTON RA, PAN X, FLANIGAN DC. Autol-ogous chondrocyte implantation: a systematicreview. J Bone Joint Surg Am 2010; 92: 2220-2233.

11) SARIS DB, VANLAUWE J, VICTOR J, HASPL M, BOHNSACK

M, FORTEMS Y, VANDEKERCKHOVE B, ALMQVIST KF, CLAES

T, HANDELBERG F, LAGAE K, VAN DER BAUWHEDE J, VAN-DENNEUCKER H, YANG KG, JELIC M, VERDONK R, VEULE-MANS N, BELLEMANS J, LUYTEN FP. Characterizedchondrocyte implantation results in better struc-tural repair when treating symptomatic cartilagedefects of the knee in a randomized controlled tri-al versus microfracture. Am J Sports Med 2008;36: 235-246.

12) VASILIADIS HS, WASIAK J. Autologous chondrocyteimplantation for full thickness articular cartilagedefects of the knee. Cochrane Database SystRev 2010; 6: CD003323.


13) BECERRA J, ANDRADES JA, GUERADO E, ZAMORA-NAVAS

P, LOPEZ-PUERTAS JM, REDDI AH. Articular Cartilage:Structure and Regeneration. Tissue Eng Part BRev 2010; 16: 617-627.

14) BRITTBERG M, LINDAHL A, NILSSON A, OHLSSON C, ISAKS-SON O, PETERSON L. Treatment of deep cartilagedefects in the knee with autologous chondrocytetransplantation. N Engl J Med 1994; 331: 889-895.

15) GIANNINI S, BUDA R, GRIGOLO B, VANNINI F, DE

FRANCESCHI L, FACCHINI A. The detached osteo-chondral fragment as a source of cells for autol-ogous chondrocyte implantation (ACI) in the an-kle joint. Osteoarthritis Cartilage 2005; 13: 601-607.

16) AKIMAU P, BHOSALE A, HARRISON PE, ROBERTS S, MC-CALL IW, RICHARDSON JB, ASHTON BA. Autologouschondrocyte implantation with bone grafting forosteochondral defect due to posttraumatic os-teonecrosis of the hip–a case report. Acta Orthop2006; 77: 333-336.

17) PETERSON L, VASILIADIS HS, BRITTBERG M, LINDAHL A.Autologous chondrocyte implantation: a long-termfollow-up. Am J Sports Med 2010; 38: 1117-1124.

18) IWASA J, ENGEBRETSEN L, SHIMA Y, OCHI M. Clinicalapplication of scaffolds for cartilage tissue engi-neering. Knee Surg Sports Traumatol Arthrosc2009; 17: 561-577.

19) HADDO O, MAHROOF S, HIGGS D, DAVID L, PRINGLE J,BAYLISS M, CANNON SR, BRIGGS TW. The use ofChondrogide membrane in autologous chondro-cyte implantation. Knee 2004; 11: 51-55.

20) KIELPINSKI G, PRINZI S, DUGUID J, DU MOULIN G.Roadmap to approval: use of an automated steril-ity test method as a lot release test for Carticel®,autologous cultured chondrocytes. Cytotherapy2005; 7: 531-541.

21) COLLINS PJ, HENNESSY LK, LEIBELT CS, ROBY RK, REEDERDJ, FOXALL PA. Developmental Validation of a Sin-gle-Tube Amplification of the 13 CODIS STRLoci, D2S1338, D19S433, and amelogenin: theAmpFSTR Identifiler PCR Amplification Kit. JForensic Sci 2004; 49: 1265-1277.

22) MULERO JJ, CHANG CW, LAGACÉ RE, WANG DY, BAS

JL, MCMAHON TP, HENNESSY LK. Development andvalidation of the AmpFlSTR MiniFiler PCR Ampli-fication Kit: a MiniSTR multiplex for the analysisof degraded and/or PCR inhibited DNA. J Foren-sic Sci 2008; 53: 838-852.

23) FOREMAN LA, EVETT IW. Statistical analyses to sup-port forensic interpretation for a new ten-locusSTR profiling system. Int J Legal Med 2001; 114:147-155.

24) PRESCIUTTINI S, CERRI N, TURRINA S, PENNATO B, ALÙ M,ASMUNDO A, BARBARO A, BOSCHI I, BUSCEMI L, CAENAZ-ZO L, CARNEVALI E, DE LEO D, DI NUNNO C, DOMENICI

R, MANISCALCO M, PELOSO G, PELOTTI S, PICCININI A,PODINI D, RICCI U, ROBINO C, SARAVO L, VERZELETTI A,VENTURI M, TAGLIABRACCI A. Validation of a largeItalian Database of 15 STR loci. Forensic Sci Int2006; 156: 266-268.

833


25) DALLARI D, FINI M, STAGNI C, TORRICELLI P, NICOLI AL-DINI N, GIAVARESI G, CENNI E, BALDINI N, CENACCHI,A, BASSI A, GIARDINO R, FORNASARI P-M, GIUNTI A.In vivo study on the healing of bone defectstreated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts,alone and in combination. J Orthop Res 2006;24: 877-888.

26) FACCHINI A, LISIGNOLI G, CRISTINO S, ROSETI L, DE

FRANCESCHI L, MARCONI E, GRIGOLO B. Human chon-drocytes and mesenchymal stem cells grown on-to engineered scaffold. Biorheology 2006; 43:471-480.

27) SCHAEFFER WI. Terminology associated with cell,tissue, and organ culture, molecular biology, andmolecular genetics. Tissue Culture AssociationTerminology Committee. In vitro Cell Dev Biol1990; 26: 97-101.

28) CABRERA CM, COBO F, NIETO A, CORTE´S JL, MONTES

RM, CATALINA P, CONCHA A. Identity tests: determi-nation of cell line cross-contamination. Cytotech-nology 2006; 51: 45-50.

29) PHUCHAREON J, OHTA Y, WOO JM, EISELE DW, TETSUO. Genetic profiling reveals cross-contamina-tion and misidentification of 6 adenoid cysticcarcinoma cell lines: ACC2, ACC3, ACCM, AC-CNS, ACCS and CAC2. PLoS One 2009; 25 4:6040.

30) GARCIA S, BERNAD A, MARTÍN MC, CIGUDOSA JC,GARCIA-CASTRO J, DE LA FUENTE R. Pitfalls in spon-taneous in vitro transformation of human mes-enchymal stem cells. Exp Cell Res 2010; 316:1648-1650.

31) DE LA FUENTE R, BERNAD A, GARCIA-CASTRO J, MARTIN

MC, CIGUDOSA JC. Retraction: Spontaneous hu-man adult stem cell transformation. Cancer Res2010; 70: 6682.

32) LINS AM, MICKA KA, SPRECHER CJ, TAYLOR JA, BACHER

JW, RABBACH DR, BEVER RA, CREACY SD, SCHUMM JW.Development and population study of an eight-lo-cus short tandem repeat (STR) multiplex system.J Forensic Sci 1998; 43: 1168-1180.

33) KRISTT D, ISRAELI M, NARINSKI R, OR H, YANIV I, STEIN J,KLEIN T. Hematopoietic chimerism monitoringbased on STRs: quantitative platform perfor-mance on sequential samples. J Biomol Tech2005; 16: 380-391.

34) BUTLER JM, SHEN Y, MCCORD BR. The developmentof reduced size STR amplicons as tools for analy-sis of degraded DNA. J Forensic Sci 2003; 48:1054-1064.

35) WIEGAND P, KLEIBER M. Less is more-length reduc-tion of STR amplicons using redesigned primers.Int J Legal Med 2001; 114: 285-287.

36) BUTLER JM. Forensic DNA Typing, Biology, Tech-nology and Genetics of STR markers. ElsevierAcademic Press (2nd Edition), New York 2005.

37) CHINNERY PF, SAMUELS DC, ELSON J, TURNBULL DM.Accumulation of mitochondrial DNA mutations inageing, cancer, and mitochondrial disease: isthere a common mechanism? Lancet 2002; 360:1323-1325.

38) JETHVA R , OTSURU S , DOMINICI M, HORWITZ EM.Cell therapy for disorders of bone. Cytotherapy2009; 11: 3-17.

pharmacol sci 2013;17:820-833 a novel dna profiling ...lized to repair or regenerate injured tissues...

Documents