B95-8 Immortalizzazione di B Linfociti

EB 

V 

EB 

V 

EB 

V 

B95-8 

EB 

V 

EB 

V 

EB 

V 

EB 

V 

EB 

V 

Linea B 

linfoblastoide 

Immortalizzazione 

di B Linfociti 

Linfocita 

B Umano 

Linea B 

linfoblastoide

Accorgimenti Tecnici per lmmortalizzare 

B-Linfociti 

• Seminare le cellule B95-8 alla concentrazione cellulare 

di 200.000 cell/ml ed incubare a 33°C per 2-3 settimane 

senza operare alcun cambio del terreno colturale 

• Il sovranatante raccolto deve essere sottoposto a 

filtrazione mediante l’utilizzo di filtri con diametro di 

0.22 µm 

• All’atto dell’infezione la concentrazione cellulare di 

cellule mononucleate deve essere di 2X10 6 /ml in un 

terreno colturale costituito dal 50% di sovranatante 

EBV positivo (derivato dalla coltura delle B95-8)

B95-8

Linfociti Umani B EBV+ 

in corso di 

stabilizzazione

Linfociti B Umani EBV+ 

stabilizzati

Linee Cellulari B Linfoblastoidi Umane 

Immortalizzate 

Linee B Linfoblastoidi Umane 

•Consentono di superare il problema inerente la 

scarsa disponibilità di campione reperibile dal 

paziente stesso 

•Rilevazione di particolari mutazioni di interesse 

diagnostico 

•Modelli di studio di particolari alleli HLA sia da 

un punto di vista molecolare che cellulare 

•Possono essere utilizzate allo scopo di migliorare 

i protocolli inerenti i controlli di qualità relativi 

alle indagini diagnostiche

Herpesvirus saimiri (HVS) 

•HVS è un γ2-herpesvirus che infetta le scimmie 

(neoplasie del lineage T); 

•HVS è in grado di infettare sia linee cellulari 

epiteliali (p.e. OMK, CHO) che linfociti T 

CD4+/CD8+; 

•HVS presenta un’elevata omologia di sequenza con 

human herpesvirus-8, HHV-8, associato al sarcoma di 

Kaposi. 

Dott. Francesca D’Alessio 

Means R.E., Virology, 2004


•Il preciso meccanismo attraverso il quale HVS entra 

all’interno delle cellule non è ancora abbastanza chiaro; 

•E’ noto che l’ingresso del virus nel compartimento 

intracellulare è preceduto dall’iniziale interazione tra le 

glicoproteine virali ed i proteoglicani cellulari stessi 

contenenti l’eparan-solfato o altre tipologie di 

glicosaminoglicani presenti sulla superficie cellulare; 

•In particolare, HHV-8 utilizza la glicoproteina k8.1 per 

interagire con i glicosaminoglicani cellulari; 

•Il gene k8.1 di HHV-8 è un omologo posizionale del gene 

Orf-51 di HVS per cui sono stati condotti diversi studi 

su tale gene e sul suo prodotto proteico. 




•Tali studi hanno dimostrato che glicoproteina virale Orf-51 

effettivamente gioca un ruolo importante nell’interazione tra HVS e 

membrana cellulare in quanto capace di legare glicosaminoglicani e 

proteoglicani contenenti eparan-solfato, espressi in grande quantità 

sulla superficie di cellule epiteliali (tipo OMK); 

•Per quanto riguarda, invece, l’infezione dei linfociti T, è noto che 

essi non presentano sulla loro membrana elevate quantità di 

proteoglicani contenenti eparan-solfato; quindi, per le interazioni 

iniziali con questa classe di cellule, potrebbero essere coinvolte altre 

proteine virali. 



HVS 

HVS 

HVS 

Procedura per infettare le 

cellule OMK 

HVS 

Le cellule OMK 

diventano un serbatoio 

per infettare i T linfociti 

HVS 

OMK

HVS HVS 

HVS 

OMK 

HVS 

HVS 

HVS 

HVS 

Linea T 

linfoblastoide 

Immortalizzazione 

di T Linfociti 

Linfocita T 

Umano 

Linea T 

linfoblastoide

Le cellule OMK

Accorgimenti tecnici per infettare 

I T-linfociti 

• E’ consigliabile utilizzare in tutti i passaggi della 

procedura d’infezione il terreno colturale costituito da 

RPMI 1640 ed uno dei terreni sintetici simili a AIM-V 

della Ditta Gibco nella proporzione di 1:1 

• Prima dell’infezione, piastrare le cellule mononucleate alla 

concentrazione di 500.000 cell/ml in presenza di PHA alla 

concentrazione di 1µg/ml ed incubare per 3 giorni 

• Seminare le cellule mononucleate alla concentrazione di 

1X10 6 cellule/ml in presenza di IL-2 alla concentrazione di 

50-100UI/ml ed 1ml di sovranatante HVS, strain C-488, 

derivato dalla lisi completa di cellule OMK; utilizzare 24wp 

• Coltivare per circa due mesi, avendo cura di operare 

regolari cambi di terreno colturale

Phytoaemoagglutinin 

(PHA) 

• Lectina mitogena ottenuta dal fagiolo 

• Agglutina gli eritrociti legandosi a specifici residui di 

carboidrati 

• Si lega ai linfociti T e B stimolandone in vitro la mitosi

Linee Cellulari T Linfoblastoidi Umane 

Immortalizzate 

Linee T Linfoblastoidi Umane 

•Modelli di studio per approfondire gli aspetti 

biologici legati all’immunodeficienza da assenza 

della catena γ del CD3 

•Modelli di studio per la sindrome di Wiskott- 

Aldrich legata ad immunodeficienza di T linfociti 

•Modelli di studio per la Sclerosi Multipla in cui 

sono coinvolti principalmente i linfociti T

Saggio di Proliferazione Cellulare 

•Effetti citotossici o inibitori sulla crescita cellulare da parte di una 

molecola 

•Effetti citotossici o citostatici sulla crescita cellulare da parte di 

farmaci antitumorali 

•Effetti citopatici sostenuti da virus sulla crescita cellulare 

•Screening di composti con potenziale attività antivirale 

•Screening di antibiotici

Saggio Colorimetrico Basato sul Reagente MTT 

Mosmann, 1983 

•La metodica è basata sull’abilità di una deidrogenasi 

mitocondriale, presente nelle cellule vitali, di intervenire 

sugli anelli di tetrazolio della molecola MTT di color giallo 

pallido 

•La reazione procede con la formazione di cristalli di 

formazano di colore blu scuro che si accumulano 

nell’ambiente intracellulare 

•I granuli possono essere solubilizzati attraverso l’uso di 

DMSO oppure Isopropanolo 0.1N in HCl e quantizzati 

mediante un saggio colorimetrico (ELISA reader)

MTT (Sale 

di 

Tetrazolio) 

Saggio Colorimetrico Basato sul Reagente MTT 

• Assorbimento delle molecole di MTT da parte delle cellule in esame 

• Metabolizzazione dell’ MTT a livello mitocondriale da parte degli enzimi 

deidrogenasi dipendenti da NADH 

•Formazione e precipitazione dei cristalli di Formazano che sono 

insolubili in soluzioni acquose. 

N 

N 

N N+ 

S 

N 

CH 3 

NADH 

CH 3 

NAD + 

N 

NH 

N 

N 

Formazano 

S 

N 

CH 3 

CH 3

SAGGIO MTT 

Sospensione 

cellulare + 

MTT 

Misurazione 

dell’assorbanza 

dei campioni 

tramite ELISA 

Reader con 

filtro da 

550nm 

INCUBAZIONE 

(3 – 4h) a 37°C 

Solubilizzazione 

dei Sali di 

formazano 

tramite 

Isopropanolo 

0,1N HCl 

oppure DMSO

Principi del saggio colorimetrico basato sul KIT ViaLight 

Il Kit si basa sulla misura della quantità di ATP presente nelle 

cellule metabolicamente attive. Il saggio sfrutta l’enzima 

Luciferasi che catalizza la formazione di luce utilizzando la 

Luciferina e l’ATP come substrati secondo la reazione: 

ATP + Luciferina + O 2 

Luciferasi 

Mg 2+ 

Ossiluciferina + 

AMP+ PPi + CO 2 + 

Luce

La Dottoressa Elisabetta Mariotti, 

Specializzanda della Scuola di 

Specializzazione in Biochimica Clinica 

della Federico II vi suggerisce queste 

slides allo scopo di poter calcolare il 

corretto numero di duplicazioni di una 

popolazione cellulare oggetto di studio. 

Ciò vi consentirà di poter stabilire se la 

coltura di interesse è una coltura 

primaria, secondaria, ecc…

Coltura a Breve Termine 

< 10 duplicazioni 

Coltura Primaria 

Coltura a Lungo Termine 

50/60 duplicazioni 

Coltura Secondaria 

Linea Cellulare Continua 

>150/200 duplicazioni 

•Fase di adattamento 

•Fase logaritmica 

•Fase di senescenza, a meno che 

•Non intervenga un fenomeno di trasformazione da cui origina una linea cellulare continua in grado di 

replicarsi indefinitamente. 

Il grafico mostra l’evoluzione del logaritmo del numero di cellule in 

funzione del tempo

CALCOLO delle DUPLICAZIONI della POPOLAZIONE 

CELLULARE in ESAME 

ovvero POPULATION DOUBLING (PD) 

[log 10 (N H) – log 10(N 1)] 

PD = ____________________ 

log 10 (2) 

N 1 = numero di cellule seminate 

N H = numero di cellule raccolte 

log 10 (2) = valore costante pari a 0,3 

Critical Parameters for the Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood. 

Stem Cells 2004; 22:625-634

100000 300000 

N° CELLULE 

SEMINATE = N 1 

48 h 

N° CELLULE 

RACCOLTE = N H 

[log10 (NH ) – log10 (N1 )] [log10 (300000) – log10(100000)] 

PD = ____________________ = ______________________ 

log10 (2) 

0,3 

300000 1000000 

N° CELLULE 

SEMINATE = N 1 

48 h 

N° CELLULE 

RACCOLTE = N H 

PD a = 1,6 

5,48 – 5 

= _________ 

0,3 

PD b = 1,73 

[Log10 (NH ) – Log10 (N1 )] [log10 (1000000) – log10(300000)] 6 – 5,48 

PD = ____________________ = ______________________ = ___________ 

Log10 (2) 

0,3 

0,3 

PD a + PD b = 1,6 + 1,73 = 3,33 

La popolazione cellulare in esame in 96 ore ha duplicato 3,33 volte 

= 1,6 

=1,73

Le Prime Linee Cellulari Continue 

Johns Hopkins 

University, 1951 

Hela 

University of 

Ibadan, 1963 

Raji

Hela

Raji

Aderenti 

Linee Cellulari Continue 

Morfologia Colturale 

Semi-aderenti 

In 

sospensione

Linee Cellulari Continue Aderenti 

Epithelial 

Epithelioid 

Fibroblast 

Fibroblastoid 

Endothelial 

Endothelial-like 

Neuronal 

Neuronal-like

Linee Cellulari 

Aderenti Epiteliali

German Collection of Microorganisms and Cell Cultures 

Dept. Human and Animal Cell Cultures 

Cell Line: P-19 

Cell Type: mouse embryonal carcinoma 

DSMZ No: ACC 316 

Origin: established from the teratocarcinoma induced in a C3H/He strain mouse 

DSMZ Cell Culture Data: 

Morphology: epitheloid cells growing as adherent monolayer 

Medium: 80-90% alpha-MEM + 10-20% FBS 

Subculture: split confluent culture 1:10 to 1:20 once or twice a week using 

trypsin/EDTA; seed out at ca. 0.3-0.4 x 105 cells/cm2 

Incubation: at 37 /C with 5% CO2 

Doubling Time: doubling time of ca. 2-3 days 

Harvest: cell harvest of max. 0.5 x 106 cells/cm2 

Storage: frozen with 70% medium, 20% FBS, 10% DMSO at about 2 x 106 

cells/ampoule 

DSMZ Scientific Data: 

Mycoplasma: negative in DAPI, microbiological culture, RNA hybridization, PCR assays 

Species: confirmed as murine with IEF of AST, LDH, MDH, NP 

Cytogenetics: murine near-diploid karyotype with 16% polyploidy; 40(31-44); one 

centric fusion marker; resembles published karyotype 

Viruses: ELISA: reverse transcriptase negative



Cell Line: HELA 

Cell Type: human cervix carcinoma 

Origin: established from the epitheloid cervix carcinoma of a 31-year-old black woman in 

1951; later diagnosis changed to adenocarcinoma; first aneuploid, continuously cultured 

human cell line 


Morphology: epithelial-like cells growing in monolayers 

Medium: 90% MEM (with Earle's salts) + 10% FBS + 2 mM L-glutamine + non-essential 

amino acids (cells also grow well in 90-95% RPMI 1640 + 5-10% FBS) 

Subculture: split confluent culture 1:4 to 1:6 every 3-5 days using trypsin/EDTA; cells 

reach confluence quickly; seed out at ca. 1-2 x 106 cells/80 cm2 


Doubling Time: doubling time of ca. 48 hours 

Harvest: cell harvest of ca. 5-15 x 106 cells/175 cm2 


cells/ampoule 



Immunology: cytokeratin+, cytokeratin-7+, cytokeratin-8+, cytokeratin-17+, cytokeratin- 

18+, desmin-,endothel-, GFAP-, HMB-45-, neurofilament-, vimentin+ 

Fingerprinting: multiplex PCR of minisatellite markers revealed a unique DNA profile 

Species: confirmed as human with IEF of G6PD, MDH, NP 

Cytogenetics: human hypertriploid/hypotetraploid karyotype with 15% polyploidy; 

82(77-84)XXX, -X, -2, -3, -4, +5, +5, +5, +5, -7, -8, -11, -13, -14, -15, -16, -17, -18, 

-19, -21, -22, +mar; der(1)t(1;3)(p11;q11), add(2)(q37), del(3)(q13), 

der(3)t(3;5)(q11;q11), i(5p)x4, del(6)(q15), der(9)t(1;9)(p11;p11), add(12)(q23), i(15q), 

der(19)t(13;19)(q22;p13)x1-2, add(21)(p11), del(?22)(q12) 

Viruses: ELISA: reverse transcriptase negative; PCR: EBV-, HBV-, HCV-, HHV-8-, 

HIV-, HTLV-I/II

ATCC 

Linee Cellulari Aderenti Endoteliali 

CPAE is an endothelial cell line derived from the main stem pulmonary 

artery of a young cow (Bos taurus). This line was initiated in January, 

1979 by P. Del Vecchio from artery lumen scrapings dispersed by 

enzyme treatment. 

Subsequent to the publication of the sixth edition of the ATCC catalog, 

tests for bovine diarrhea virus (BVD) have indicated that CPAE cells test 

positive for BVD viral antigen 

Medium: Minimum essential medium (Eagle) with 2 mM L-glutamine, 0.1 

mM non-essential amino acids, and 1.0 mM sodium pyruvate, 80%; fetal 

bovine serum, 20% 

Remove medium, and rinse with 0.25% trypsin, 0.03% EDTA solution. 

Remove the solution and add an additional 1 to 2 ml of trypsin-EDTA 

solution. Allow the flask to sit at room temperature (or at 37C) until the 

cells detach. 

Freeze medium: culture medium 95%; DMSO, 5%


Aderenti 

Fibroblastoidi



Cell Line: COS-1 

Cell Type: African green monkey kidney 

Origin: established from CV-1 Simian cells (cercopithecus aethiops) which were 

transformed by an origin-defective mutant of SV-40; cells are host cells for the 

propagation of pure populations of recombinant SV virus; classified as risk category 1 

according to the German Central Commission for Biological Safety (ZKBS) 


Morphology: fibroblast-like, adherent monolayers 

Medium: 90% Dulbecco's MEM + 10% FBS 

Subculture: split confluent cultures 1:3 to 1:10 every 3-7 days using trypsin/EDTA; seed 

out at ca. 2 x 106 cells/80 cm2 in 10 ml medium 



Harvest: about 5 x 106 cells/80 cm2 


cells/ampoule 



Fingerprinting: unique Hinf I-(gtg)5 DNA profile (identical to COS-7, DSM ACC 60) 

Species: confirmed as of non-human primate origin with IEF of AST, NP, PEP B 

Cytogenetics: hypotetraploid simian karyotype with 4% polyploidy; 102(95-108); 

structural rearrangements present 

Viruses: ELISA: reverse transcriptase negative; PCR: EBV-, HCV-, HBV-, HIV-, 

HTLV-I/II


Aderenti Neuronali



Cell Line: NEURO-2A 

Cell Type: mouse neuroblastoma 

Origin: established from the spontaneous tumor of a strain A albino mouse 


Morphology: about 30% of the cells grow like neuronal cells; 70% are round, loosely 

attached cells 

Medium: 90% Dulbecco's MEM + 10% FBS + 1x non-essential amino acids 

Subculture: split culture 1:4 about once a week using trypsin/EDTA, cells do not grow to 

confluence and grow rather slowly; seed out at ca. 1 x 106 cells/25 cm2 



Harvest: about 8 x 106 cells/75 cm2; maximal density at ca. 20 x 106 cells/175 cm2 


cells/ampoule 



Species: confirmed as mouse with IEF of AST, MDH, NP, PEP B 

Viruses: ELISA: reverse transcriptase positive


Continue 

Semi-Aderenti



Cell Line: U-266 

Cell Type: human multiple myeloma 

Origin: established from the peripheral blood of a 53-year-old man with IgE-secreting 

myeloma (refractory, terminal) in 1968; cells were described to produce IgE lambda; 

possible fusion partner for hybridoma production; cells express mRNA for bcl-2 gene 


Morphology: round to polygonal, single or clustered cells in suspension, some loosely 

adherent 

Medium: 90% RPMI 1640 + 10% FBS 

Subculture: split saturated culture 1:2 to 1:4 every 2-3 days; maintain at 0.2-1.0 x 106 

cells/ml; cells grow partially (loosely) adherent and can be removed with a bent Pasteur 

pipette (trypsin is not necessary); seed out at ca. 0.5-0.7 x 106 cells/ml 



Harvest: saturation density at 1.0-1.5 x 106 cells/ml 


cells/ampoule 


Mycoplasma: contamination was eliminated with Ciprobay (ciprofloxacin), then negative 

in DAPI, microbiological culture, PCR assays 

Immunology: CD3-, CD4-, CD5-, CD10-, CD13-, CD14-, CD15-, CD19-, CD21-, CD22- 

, CD33+, CD34-, CD37-, CD38(+), CD138+, HLA-DR+, sm/cyIgG-, sm/cyIgM-, 

sm/cykappa-, smlambda-, cylambda+ 


Species: confirmed as human with IEF of AST, MDH, NP 

Cytogenetics: human complex hypodiploid karyotype with 6.5% polyploidy; 44(40- 

46)XY, -8, -10, -13, -15, +2mar, t(1;11)(p33;q13), add(3)(q27), t(4;11)(q?21;q23), 

add(7)(q32), add(8)(q24), add(9)(q34), add(10)(p14), add(14)(p11), add(17)(p11), 

add(18)(p12), der(22)t(15;22)(q21;q13); 11q13 breakpoint recurrent in multiple myeloma 

Viruses: ELISA: reverse transcriptase negative; PCR: EBV-, HBV-, HCV-, HIV-, 

HTLV-I/II



Cell Line: CCRF-CEM 

Cell Type: human T cell leukemia 

Origin: established from the peripheral blood of a 3-year-old Caucasian girl with acute 

lymphoblastic leukemia (ALL) at relapse (terminal) in 1964 (first continuous human T- 

ALL cell line) 


Morphology: round cells growing singly in suspension and as adherent epitheloid cells 

forming a monolayer 


Subculture: split saturated culture every 2-4 days; cells form monolayers until confluence 

and will then go into suspension; after transfer of suspension cells to a new flask, a new 

monolayer will develop; it may be necessary to use trypsin/EDTA or vigorous pipetting 

to detach the adherent cells; seed out after thawing at 0.5-1.0 x 106 cells/ml 


Doubling Time: doubling time of ca. 24-30 hours 

Harvest: maximal density at ca. 2 x 106 cells/ml (cells in suspension) or 4-5 x 106 

cells/80 cm2 flask 

Storage: frozen with 70% medium, 20% FBS, 10% DMSO at about 4-6 x 106 

cells/ampoule 


Mycoplasma: contamination was eliminated with Mycoplasma Removal Agent, then 

negative in DAPI, microbiological culture, RNA hybridization, PCR assays 

Immunology: CD2-, CD3+, CD4+, CD5+, CD6+, CD7+, CD8-, CD13-, CD14-, CD15+, 

CD19-, CD33-, CD34-, CD68-, CDw90-, HLA-DR-, TCRalpha/beta+, 

TCRgamma/delta- 


Species: confirmed as human with IEF of LDH, NP 

Cytogenetics: human near-tetraploid karyotype with extensive subclonal variation; 90(88- 

101)XX, -X, -X, +20, +20, t(8;9)(p11;p24)x2, der(9)del(9)(p21-22)del(9)(q11q13- 

21)x2; sideline with +5, +21, add(13)(q3?3), del(16)(q12) 

Viruses: ELISA: reverse transcriptase negative; PCR: EBV-, HBV-, HCV-, HIV-, 

HTLV-I/II

Linee Cellulari Continue in Sospensione 

• a singole cellule 

• a piccoli clumps 

• a grandi clumps

A Singole Cellule



Cell Line: BV-173 

Cell Type: human B cell precursor leukemia 

Origin: established from the peripheral blood of a 45-year-old man with chronic myeloid 

leukemia (CML) in blast crisis in 1980; contains the t(9;22) b2-a2 fusion gene 


Morphology: round to elongated, single cells in suspension 


Subculture: cells are difficult to culture! cells appear to grow better in 24-well-plates than 

in flasks; maintain at 0.5-1.0 x 106 cells/ml; cells grow slowly, split 1:2 to 1:3 every 3-4 

days; seed out at ca. 1 x 106 cells/ml 



Harvest: maximum density at 2-3 x 106 cells/ml 


cells/ampoule 



Immunology: CD3-, CD10+, CD13+, CD19+, CD20+, CD34-, CD37-, CD79a-, 

cyCD79a+, CD80-, CD138-, HLA-DR+, sm/cyIgG-, sm/cyIgM-, sm/cykappa-, 

sm/cylambda- 


Species: confirmed as human with IEF of G6PD, MDH, NP 

Cytogenetics: human hyperdiploid karyotype; 47(46-48)X/XY, -9, +22, +mar, 

add(1)(q42), add(8)(p23), t(9;22)(q34;q11), der(22)t(9;22)(q34;q11), der(?)t(9;?)(?p11;?); 

resembles published karyotype 


HIV-, HTLV-I/II



Cell Line: K-562 

Cell Type: human chronic myeloid leukemia in blast crisis 

Origin: established from the pleural effusion of a 53-year-old woman with chronic 

myeloid leukemia (CML) in blast crisis in 1970; cells can be used as highly sensitive 

targets in in-vitro natural killer assays; cells produce hemoglobin; cells carry the 

Philadelphia chromosome with a b3-a2 fusion gene 


Morphology: round large, single cells in suspension 


Subculture: maintain at 0.1-0.5 x 106 cells/ml; split 1:3 to 1:5 every 3 days; seed out at 

ca. 0.3-0.5 x 106 cells/ml 



Harvest: maximal density at 1.0-1.5 x 106 cells/ml 

Storage: frozen with 70% medium, 20% FBS, 10% DMSO at about 3-5 x 106 

cells/ampoule 


Mycoplasma: contamination was eliminated with Ciprobay (ciprofloxacin), then negative 

in DAPI, microbiological culture, PCR assays 

Immunology: CD3-, CD13+, CD19-, CD34-, CD41(+), CD42+, CD45+, CD71+, 

GlyA(+) 


Species: confirmed as human with IEF of AST, MDH, NP 

Cytogenetics: human hypotriploid karyotype without sharp mode; 61-68XX, -X, -3, 

+7, -13, -18, +3mar, del(9)(p11/13), der(14)t(14;?)(p11;?), der(17)t(17;?)(p11/13;?), 

der(?18)t(15;?18)(q21;?q12), 

del(X)(p22); two markers appear from FISH to have arisen from Ph 


HIV-, HTLV-I/II



Cell Line: NB-4 

Cell Type: human acute promyelocytic leukemia 

Origin: established from the bone marrow of a 23-year-old woman with acute 

promyelocytic leukemia (APL = AML FAB M3) in second relapse in 1989; patented cell 

line; cells carry the t(15;17) PML-RARA fusion gene 


Morphology: single cells in suspension, round and polymorphic cells 


Subculture: maintain at 0.5-1.0 x 106 cells/ml; optimal split ratio at 1:2 to 1:3 every 2-3 

days; seed out at ca. 0.5-1 x 106 cells/ml; viability may be low after thawing, but cells 

will recover soon when cultured initially with 20% FBS; cells might also grow better in 

24-well-plates than in culture flasks 



Harvest: cell harvest of 1.5-2.0 x 106 cells/ml 


cells/ampoule 


Mycoplasma: contamination was eliminated with Mycoplasma Removal Agent, then 

negative in DAPI, microbiological culture, RNA hybridization, PCR assays 

Immunology: CD2-, CD3-, CD4+, CD5-, CD6-, CD7-, CD8-, CD11b-, CD13+, CD14-, 

CD15+, CD19-, CD33+, CD34-, CD38+, CD68-, HLA-DR-, TCRalpha/beta-, 

TCRgamma/delta- 


Species: confirmed as human with IEF of AST, MDH 

Cytogenetics: human hypertriploid karyotype with 3% polyploidy; 78(71-81)XX, - 

X, +2, +6, +7, +7, +11, +12, +13, +14, +17, -19, +20, +4mar, der(8)t(8;?)(q24;?), 

der(11)t(11;?)(?->::11p15->11q22.1::11q13->22.1:), der(12)t(12;?)(p11;?), 14p+, 

t(15;17)(q22;q11-12.1), der(19)t(10;19)(q21.1;p13.3)x2; identity confirmed 


HIV-, HTLV-I/II

A Piccoli Clumps



Cell Line: KASUMI-1 

Cell Type: human acute myeloid leukemia 

Origin: established from the peripheral blood of a 7-year-old Japanese boy with acute 

myeloid leukemia (AML FAB M2) (in 2nd relapse after bone marrow transplantation) in 

1989; cells carry the t(8;21) ETO-AML1 fusion gene 


Morphology: round cells growing singly or in small clumps in suspension 


Subculture: split saturated culture about 1:2 to 1:3 every 3 days; optimal density at about 

0.5 x 106 cells/ml; viability may drop below 50% within the first 24 hours after thawing; 

we recommend to start culture with a cell concentration of >1 x 106 cells/ml and to 

cultivate initially in 24-well plates in the presence of 20% FBS; a significant amount of 

cell debris is always visible in the background which might be due to granula or other 

intracellular material 



Harvest: saturation density at about 1.0 x 106 cells/ml 


cells/ampoule 



Immunology: CD3-, CD4+, CD13+, CD14-, CD15+, CD19-, CD33+, CD34+, CD38+, 

CD68-, CD71+, HLA-DR(+) 


Species: confirmed as human with IEF of AST, PEP B 

Cytogenetics: human hypodiploid karyotype; 45X, -Y, -9, -13, -16, +3mar, 

t(8;21)(q22;q22), der(9)t(9;?)(p22;?), der(15)t(?9;15)((?q11;?p11); carries both partners 

of 8;21 translocation associated with AML (mainly FAB M2) 

Viruses: ELISA: reverse transcriptase positive; PCR: EBV-, HBV-, HCV-, HHV-8-, 

HIV-, HTLV-I/II

A Grandi Clumps: 

il caso delle PC-12



Cell Line: PC-12 

Cell Type: rat adrenal pheochromocytoma 

Origin: established from a transplantable rat adrenal pheochromocytoma in 1976; cells 

were described to synthesize catecholamines (dopamine, norepinephrine); in response to 

nerve growth factor (NGF) a neuronal phenotype could be induced reversibly 


Morphology: small cells growing in clumps in suspension, adhering poorly to plastic 

Medium: 85% RPMI 1640 + 10% horse serum + 5% FBS 

Subculture: split saturated culture 1:2 to 1:3 every 3-4 days; cells grow in suspension 

without collagen; using collagen (Collagen S- Roche at 30 microg/ml) cells grow 

adherent, faster and to a higher density; the adherent cells may be detached mechanically 

without trypsin, for single cells the suspension should be passed several times through a 

syringe with a 22-gauge needle; seed out at ca. 5-15 x 106 cells/25 cm2 



Harvest: about 1-2 x 106 cells/ml or 30 x 106 cells/80 cm2 


cells/ampoule 



Species: confirmed as rat with IEF of AST, MDH, NP, PEP B 

Cytogenetics: rat hypodiploid karyotype with 20% polyploidy; 39(38-40) 

Viruses: ELISA: reverse transcriptase negative

I due più grandi problemi di una 

Cell Line Culture 

• Cross-Contaminazione 

• Contaminazione da Micoplasma

Cross-Contaminazione 

Una linea cellulare continua cross- 

contaminata non rappresenta più un 

modello di studio per comprendere la 

biologia delle cellule tumorali oggetto 

del nostro interesse

B95-8 Immortalizzazione di B Linfociti

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