[CANCl!ll RESEARCH 48, 589-601, Felnuy I, 1981)
`
`Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a
`
`
`
`
`Microculture Tetrazolium Assay1
`
`L. Hursey, Maciej J. C7.enrinski,
`Anne Monks, Miriam
`Michael C. Alley,2 Dominic A. Scudiero,
`Betty J. Abbott, Joseph G. Mayo, Robert H. Shoemaker, and Michael R. Boyd
`hopu,t RacMrnw,
`I&, Natimlal Quar /ll8lillde-Fmlmck
`Quar b#attl, Fadlity, FmJmdc,
`Mary/olul 21101 [M. C. ..4., D • ..4. S., ..4. M., M. L H., M. I. C.,
`Dirilion o/Ourcff �. NotimuJI Quar lruti-, B«llada, Mary/olul 20892 (B. J. A., I. G. M., R. H. S.,
`D. L F.J and �Illa/
`Tllm,pntia l'rrlpun,
`M.R.B.J
`
`Donald L. Fine,
`
`ABSTRACT
`
`METIIODS
`
`Oyopresei ratioa, aad Characterization
`
`panels of multiple well-cbaracteri7.ed human tumor cell lines.
`
`In addition, the proposed in vitro component differs from other
`dlacoffl'Y aad
`For tbe pat 30 years strmaies for tbe pnclblical
`
`in ,itro screening methodologies, the human tumor colony
`e.g.,
`dffelopmeat of potmdaJ udcanc:er ....
`baft ._ llued laqely apon
`formation assay (10, 11) which is limited by its labor-intensive
`tbe testma of aaeats in mice bearinl tnlllplaatable
`leakemils aad IOJid
`
`
`nature and the more limited range of tumor types amenable to
`tumonderiftd
`fiuma limlted naniberofmarlaea well a lumma lOUl'Cel.
`soft-agar culture.
`Tbe feulblllty of inlpie-tiaa u alteraate approacb, .-ty combined
`To determine whether such an in vitro/in vivo disease-ori­
`iaritro/ia ri,o----lorlllecd.ec,totoxldty111110111paaelsofh­
`
`
`ented screening program is feasi"ble, we have examined a num­
`tamor cell liaes deriftd fium ..... lpectnm of ...... lOlid hllllorl ii
`
`
`ber of technical questions which are fundamental to in vitro
`IIDller lnftldpdGn. A fP'OIIP of 30 cell 11w acqaired fium a 'fUiety of
`IOUffl!I aad 1eprwatiaa 8 hmg cancer
`
`
`.-thoJoales a well a 76 cell assay systems. In this report we have assessed
`whether it is
`liaes repreNlldaa
`10 other catepies of h- cancer (can:laomas of
`
`
`
`possible to cultivate a multiplicity of human tumor cell lines
`co1oa, breut, kidney, prol1ate, OYU"J, head aad neck; &lioDa; leukemia;
`
`
`under similar culture and assay conditions; whether a colori­
`melaaoaa; aad -> ba.e exhibited accepCable powth chancterls­
`
`
`for the measurement of metric assay (e.g., 12, 13) is suitable
`tb ... llllltallle CDlorlamic profiles In
`......... studanl caltare -­
`
`
`cell line growth inlu"bition; and whether individual cell lines
`by cell­
`diam. � of ill
`ritro powth In mlaucaltare well
`
`
`
`exhi"bit stable and reproduci"ble drug sensitivity profiles over
`(0.89 <
`mediated redacdoa of tetruolima allowed excelleat c:orreladoa
`time.
`of ce11ur protein m adherent ce11 Hae
`r < us> with --
`ca1tares a well a riable cell COUDt In saspemloa cell llae ca1tares (0.94
`< r < 0.99). Slace the ada-oc:ultare
`tetruollam -y pnmdes seasidl'e
`MATERIAI.S AND
`aad repredacible
`bidlees of powth a well a dna seasitiYlty la ladMdaal
`Cell Line Expwioa,
`cell liaes OYel' the - of maldple ,...... aad seTerlll IIIOllths
`'
`caltltatioll, it appean nitable for lnitlaHCaae ia ritro dna screem111-
`
`maay human
`Thus far, more tbaa 100 tumor cell lines representing
`solid tumor types have been acquired from several soun:es followiaa
`
`various methods of isolation and cultivation. Individual cell lines were
`initially photographed, expanded (two passages maximum) and cryo­
`pn:served (master stoclu) with arowth medium and SPiit-ratios recom­
`A new anticancer drug screening program based upon the use
`mended by their respective soun:es.
`Only cell lines clocumented to be
`of multiple panels of human solid tumor cell lines is under
`free of adventitious bacteria and pathogenic viruses (NCI-FCRF3 Di­
`'s Devel­
`
`development by the U. S. National Cancer Institute
`agnostic Microbiology Lab and Animal Health Diagnostic Lab) were
`
`
`
`opmental Therapeutics Program, Division of Cancer Treatment
`accepted for subsequent chancterization. Following recovery of mater
`
`(1-7). The goal of the new program is to evaluate experimental
`stocks, cell lines were adapted to a sin&le, standard culture medium:
`agents against groups of cell line panels each representing a
`RPMI 1640 (Quality Biologicals, Inc., Gaithersburg, MD) supple­
`
`major clinical category of human malignancy. Each panel (e.g.,
`mented with 10% heat-inactivated fetal bovine serum (Sterile Systems
`
`lung, colon, melanoma, renal, ovarian, and central nervous
`Hyclone, Logan, UT) and 2 mM L-glutamine (NCI-FCRF Central
`
`
`system) is to contain multiple, representative human tumor cell
`Medium Laboratory) without antilriotics and cultured under conven­
`tional culture conditions, that is, 37•c, 5% CCh, 95% air, 100% relative
`lines. Agents showing differential or selective patterns of in
`
`humidity. Cell lines were then expanded (five passages maximum) and
`
`
`vitro growth inhibition will be evaluated subsequently in
`cryopreserved
`for generation of seed stocks. Established adherent cell
`athymic mice bearing the same human tumor cell lines found
`monolayers approaching 80% confluency were harvested
`with tryplin/
`sensitive in vitro. This in vitro/in vivo approach differs from
`EDT A (NCI-FCRF Central Medium Laboratory) whereas 10me early
`previous in vivo screening programs (8, 9), which most recently
`passage adherent cell lines were harvested with solution A and 2x­
`
`consisted of a murine leukemia prescreen followed by a battery
`crystali7.ed trypSin
`Co.) according to the protocol
`m (Sigma Chemical
`
`of tests including several murine tumor models and three hu­
`
`of Shipley and Ham (14). Leukemia cell lines were subcultured by
`
`man tumor xenografts. in two fundamental ways: (a) a single
`trituration and dilution. Smsll cell lung c:arcinoma cell lines (which
`
`in vi,o murine leukemia prescreen step replaced by broad-based
`geaerally form large aareptes ia suspeasion under conventional cul­
`
`in vitro evaluations among a wide variety of cell lines; and (b)
`ture conditions) were cultured and assayed ia suspension a well a
`adherent monolayers utilizing poly-L-lysiae pretreatment
`of culture
`
`the major clinical forms of human solid tumors represented by
`vessels (15). Following recovery of seed stock, cell lines were subjected
`to isoenzyme
`and drug sensitivity
`analysis u well as preliminary arowth
`assays using one or more in vitro growth inhibition assays (described
`criteria (mycoplasma-
`below). Cell lines meeting basic quality assurance
`
`INTRODUCOON
`
`, 10/6/87;
`accepted 11/2/87.
`Recemcl 2 /2/87; reYiBed 6/18/87
`The com of publicatloD of this article - de&ayed in put by the payment
`in
`of pqe cbaqes. This artide must therefon be hereby marked IIIINrtiaemat
`Section 1734 aolely to indicate this fact.
`� with 18 U.S.C.
`1 Supported by National C- Jnstitute contnct
`NOI-C0-23910 with Pro­
`pam RelOIIRleS, Inc. The c:ontents of this publication do not necaauily rdlect
`the Yiews CB" policies of the Department of Heslth and Humu Senices nor does
`mention of trade na-, CXlllllllfflW products,
`or orpnizations
`imply endone­
`ment by the u. s. Gcnwnment.
`PDRG, Demopmental
`• To whom reci-tsforrepriatsllhouldbelldclr-.lat:
`R-11
`Institute-Frederick C-
`Therapeutics Proanm,
`Natioul C-
`FacDlty, Building 560, Room 32-60, Frederick, MD 21701.
`
`• The ablnYiations ued are: NCI-FCRF, Nadoual C- Institute-Frederick
`C- Raean:b Facility; DMSO, dimethyl sulfollide; INT, 2-q,iodonitro­
`pbenyl) -].JHlitrophenyt-5-
`pbenyl tetruoHum chloride; MCPA, microculture
`eel·
`lular protein 1111111T, MTA, mil:roc:ulture tetruollum .-y; M'JT , 3-(4,5-di­
`
`methyltbiazol•2•yl)-2,5-diphenyltelr8Zolium bromide; NBT, 2,2' -di-p-nitrophen­
`
`
`
`
`
`yl-5,5 • -diphenyl-3,3 • -(3,3' -dimethoxy-4,4' -diphenylene)ditetrazolium
`chloride; JC.., 50% of control powth absorbance.
`589
`
`

`

`MICROCULTURE TETRAZOLIUM ASSAY
`
`negative, MAP-negative, human isoenzymes only) and exhibiting suit
`able growth profiles were expanded (live serial passages from seed
`stock, maximum)
`and cryopreserved as a large number of aliquots
`designated working seed stock. Cell cryopreservation was achieved
`using a CryoMed controller
`(Model 801) and a CryoMed freezing
`chamber (No. 2700) with a step rate of -l°C/min followed by storage
`in vapor-phase liquid N2 (NCI-FCRF Central Repository). Cell
`line
`seed stocks were tested also for in vivo tumorigenicity (s.c. and i.p.
`inoculations)
`in accordance with established protocols
`(16). Cell lines
`recovered from working seed stocks were subjected to repeat myco-
`plasma tests and to more extensive in vitro growth characterization.
`Cell lines subsequently were evaluated with respect
`to stability in drug
`sensitivity profiles over the course of 20 weekly passages. In addition,
`each cell line was expanded (eight passages, maximum from seed stock
`thaw) and cryopreserved as a large number of aliquots ("roller bottle"
`stock) for in vivo characterization and assay development.
`
`Reagents
`
`reagents were purchased from Sigma Chem
`Tetrazolium/formazan
`ical Co. (St. Louis, MO): MTT (M2128), MTT formazan (M2003),
`INT (18377), INT formazan (17375), and NBT (N6876). DMSO was
`purchased from Sigma Chemical Co. (D5879), J. T. Baker Chemical
`Co. (9194-3, Phillipsburg, NJ), and American Burdick and Jackson
`Laboratories (Spectrophotometric Grade Product 081, Muskegan, MI).
`These chemicals were stored in unopened bottles at room temperature
`in the dark or in SO-ml sterile plastic tubes at —20°Cin the dark.
`Anhydrous
`isopropanol
`(505-7) and propylene glycol (P-1009) were
`purchased from Sigma Chemical Co. Reagent grade hydrochloric acid
`(A-744) and hexane (H-302-1) were purchased from Fisher Scientific
`Co. Dimethylformamide
`(27,054-7) was purchased from Aldrich Chem
`ical Co. (Milwaukee, WI) Propanol
`(spectrophotometric
`grade 9068-1)
`was purchased from American Burdick and Jackson Laboratories.
`All chemotherapeutic
`agents were obtained from the Drug Synthesis
`and Chemistry Branch, Developmental Therapeutics Program, Divi
`sion of Cancer Treatment, National Cancer Institute. Crystalline stock
`materials were stored at -20°C. Solvent-reconstituted
`chemotherapeu
`tic agents were prepared at high concentration, partitioned into multiple
`aliquots, and stored at —70°C.Just prior
`to culture application the
`contents of frozen vials were thawed and mixed. Measured aliquots
`(20-200 id) were transferred by micropipet
`(Gilson Pipetman, Models
`P200 and P1000) equipped with polypropylene tips to standard culture
`medium within polypropylene
`tubes (Sarstedt 62.554/002
`or Falcon
`2098) and serially diluted in culture medium containing an appropriate
`concentration of vehicle.
`
`In Vitro Growth/Growth Inhibition Assays
`
`Microculture Tetrazolium Assay. The methodology described below
`represents
`a modification
`of the original MTT colorimetrie
`assay
`described by Mosmann (12): In principle,
`the viable cell number/well
`is directly proportional
`to the production of formazan, which following
`solubilization, can be measured spectrophotometrically. Our modifica
`tion of the original
`in vitro assay procedures has been previously
`described (6, 17). In brief, cells were harvested from exponential-phase
`maintenance cultures (T 75 cm2 flasks; Falcon Plastics 3023), counted
`by trypan blue exclusion, and dispensed within replicate 46-well culture
`plates (Falcon Plastics 3075) in 100-¿ilvolumes using a repeating pipet
`(Eppendorf repeater 4780) or multichannel pipet (Flow Labs, Titertek).
`Following a 24-h incubation at 37°C,5% CO2, 100% relative humidity
`(Heraeus B5060EKO2 incubators or NAPCO 5300 incubators), 100 /il
`of culture medium, culture medium containing drug or culture medium
`containing drug vehicle was dispensed within appropriate wells (vehicle
`control group, N = 6; each drug treatment group, N = 3). Peripheral
`wells of each plate (lacking cells) were utilized for drug blank (N = 2)
`and medium/tetrazolium reagent blank (N = 6) "background" deter
`minations. Culture plates were then incubated for 1 to 11 days prior to
`the addition of tetrazolium reagent. MTT stock solution was prepared
`as follows: 5 mg MTT/ml PBS (Quality Biologicals, Inc.) was sterile
`filtered with 0.45-^m filter units (Nalgene type SCN) and stored at 4°C
`for a maximum of 1 month. MTT working solution was prepared just
`
`prior to culture application by diluting MTT stock solution 1:5 (v/v) in
`prewarmed standard culture medium. Alternatively, other tetrazolium
`reagents (namely, INT or NBT) were prepared and utilized in a similar
`fashion for selected experiments. Under standard MTA conditions 50
`ti\ of MTT working solution was added to each culture well (resulting
`in 50 itg MTT/250 pi total medium volume) and cultures were incu
`bated at 37°Cfor 4 to 24 h depending upon individual cell
`line
`requirements. Following incubation cell monolayers and formazan were
`inspected microscopically: Culture plates containing suspension lines
`or any detached cells were centrifuged at low speed for 5 min. All but
`10-20 M' of culture medium supernatant was removed from wells by
`slow aspiration through a blunt 18-gauge needle and replaced with 150
`n\ of DMSO (Burdick & Jackson) using a multichannel pipet. Following
`thorough formazan solubilization (trituration by pipet or vibration on
`a plate shaker),
`the absorbance of each well was measured using a
`microculture plate reader (Dynatech MR600; Alexandria, VA) at 540
`nm (single wavelength, calibration factor = 1.00) interfaced with an
`Apple He computer. Subsequently, data were stored and analyzed
`through use of Apple Soft, Apple Turnover,
`and Lotus Symphony
`software. Cell
`line growth and growth inhibition were expressed in
`terms of mean (±1SD) absorbance units and/or percentage of control
`absorbance (±1SD%) following subtraction of mean "background"
`absorbance. Linearity and reproducibility of instrument measurements
`were verified by the use of formazan reagents in appropriate
`solvent
`systems.
`Absorption spectra of formazan reagents as well as cell-generated
`formazans were measured with a UV/visible scanning spectrophotom-
`eter (Perkin-Elmer Lambda V; Perkin-Elmer Corp., Norwalk, CT).
`Samples were placed in 1-cm pathlength disposable polystyrene cuvets
`(Fisher Scientific Co. 14-385-942) except
`those solubilized in dimeth-
`ylformamide solvent which were evaluated in 1-cm pathlength glass
`spectrophotometer
`cells (Coleman S7300-4). Freshly prepared material
`was analyzed in dual beam mode with 2-nm slit width, at 120 nm/min,
`0.02 A threshold, and 0.5 s response. Instrument wavelength calibration
`was verified by examination of deuterium emission spectra to be 653.1
`±0.3 nm.
`Microculture Cellular Protein Assay. A cellular protein assay previ
`ously described by Finlay, Baguley, and Wilson (13) was adapted to the
`measurement of cell
`line growth under
`the same culture conditions
`described above for the MTA. Following 1-11 days' incubation, super
`natant culture medium was removed and 200 n\ of méthylèneblue
`(Sigma MB-1) solution [5 g/liter
`in ethanol:water
`(50%, v/v)] was
`added without delay. Following incubation at room temperature for 45
`min, unbound stain was removed by plate inversion on absorbant paper
`and subsequent emersion/dilution
`in four, 1-liter washes with distilled
`deionized water. Bound protein stain was solubilized by the addition of
`100 n\ SDS (Sigma L4509) solution (1%, v/v in water) to each well.
`Absorbances of wells were measured at 630 nm (single wavelength,
`calibration factor = 1.00) using equipment and computerized analysis
`procedures described above for the MTA.
`
`RESULTS
`
`and Cryopreservation. A
`Cell Line Acquisition, Adaptation,
`key question concerning the use of in vitro cell lines for com
`parative drug evaluation has been whether a wide variety of cell
`lines would exhibit stable growth and drug sensitivity profiles
`over serial passage. To examine this question at a practical
`level
`we have evaluated the performance of multiple cell lines which
`represent common human solid tumor malignancies.
`To date 111 cell lines derived from 10 major categories of
`human cancer (carcinomas of lung, colon, kidney, ovary, pros
`tate, and head and neck; glioma;
`leukemia; melanoma;
`and
`sarcoma)
`isolated by a variety of in vivo as well as in vitro
`techniques have been acquired, cultivated, cryopreserved,
`and
`tested. With the exception of two early passage colon adeno-
`carcinoma lines and one lung adenosquamous
`line which exhibit
`doubling times exceeding 120 h in our standard culture medium
`
`590
`
`

`

`MICROCULTURE TETRAZOLIUM ASSAY
`
`slowly in their
`(each of which also proliferates
`formulation
`respective recommended
`culture medium) all tumor cell lines
`tested to date show suitable growth under maintenance
`(T-75
`cm2 flask) as well as assay conditions
`(96-well plate). In addi
`tion,
`two fibroblast cell lines (which exhibit acceptable rates of
`growth) metabolized MTT at
`levels which are adequate
`for
`detection but which may not be desirable for screening (<0.500
`absorbance units/confluent monolayer). A total of 106 cell lines
`which meet basic quality assurance criteria and which exhibit
`suitable growth and colorimetrie profiles are listed in Table 1.
`Despite the fact that
`these cell lines have been cultivated under
`a variety of conditions
`(including 31 different culture medium
`formulations)
`in other
`laboratories,
`each cell
`line appears
`to
`have adapted adequately to one set of culture conditions
`(and a
`single, standard culture medium) as indicated by exponential
`or near-exponential
`growth following inoculation at reasonable
`cell densities (< 10,000 cells/well).
`experiments
`Microculture Tetrazolium Assay. Preliminary
`using the original MTT colorimetrie
`assay (6) revealed an
`apparently
`inadequate
`level of formazan generation by some
`cell lines, limited solubility and stability of MTT formazan, and
`an incompatability
`of the acid/isopropanol
`solvent system with
`the evaluation of some synthetic agents. Microscopic inspection
`of plates
`following tetrazolium metabolism (prior
`to solvent
`addition)
`revealed that
`these problems generally were not due
`to the ability of cells to metabolize MTT. Some cell lines such
`as NCI-H460 produced copious amounts of formazan much of
`which was insoluble in acid/isopropanol;
`other cell lines such
`as NCI-H322 and P388 exhibited significant
`formazan micro
`scopically but
`low absorbance
`readings, again due to limited
`solubility of cell-generated formazan.
`formazan sol
`These findings prompted assessment of other
`vent systems. Solubility testing and spectral analysis with a
`number of solvents including DMF, DMSO, hexane, and pro-
`pylene glycol showed that DMSO was the most suitable solvent
`for culture-generated MTT formazan as well as INT formazan;
`NBT formazan was not appreciably soluble in any of these neat
`solvents at room temperature. Microscopic
`inspection of cell
`culture plates revealed that formazans are rapidly mobilized by
`DMSO from sites within thick cell layers. Spectrophotometric
`analyzes indicate that
`the resulting DMSO/formazan
`solutions
`are stable and exhibit prominent
`absorbance in the visible light
`region (Fig. IB). While MTT formazan reagent
`is in fact totally
`soluble in anhydrous
`isopropanol
`at a concentration
`of 5 mg/
`ml, in the presence of 0.04 N HCl/isopropanol
`as specified by
`the original procedure
`(12), MTT formazan exhibits a very
`blunted absorbance at 570 nm and increased absorbance at 420
`and 300 nm (see Fig.
`\D). Color "fading" was accompanied by
`a rapid,
`irreversible shift in absorbance maximum and appeared
`to be a direct consequence of medium acidification. The absor
`bance of MTT formazan reagent
`in DMSO is approximately
`1.3x that of formazan in neat
`isopropanol
`(Fig.
`If) and more
`than 6.2x that observed in the acid/isopropanol
`solvent system.
`While the extinction coefficient of MTT formazan reagent
`in
`dimethylformamide
`(£513= 18,100 vT1 cm"1) was the highest
`
`\A and Table 2), dimethylform
`of all neat solvents tested (Fig.
`amide is not compatible with polystyrene culture vessels. On
`the grounds of improved solubility and stability of culture-
`generated MTT formazan in DMSO,
`this solvent was adopted
`in our current microculture tetrazolium assay.
`Spectral characteristics of reagent as well as culture-generated
`MTT formazan depend not only upon the organic solvent but
`also upon the presence or absence of serum. As shown in Fig.
`JA,
`the absorption spectra of culture-derived MTT formazan
`
`and MTT formazan reagent differ significantly. The former
`exhibits an absorbance maximum of 553 nm (half-height band
`width of 110 nm), whereas
`the later exhibits an absorbance
`maximum of 506 nm (half-height bandwidth of 170 nm). In the
`presence of DMSO and low serum concentration MTT for-
`mazan reagent exhibits a shift in the absorption maximum from
`506 to 553 nm and a narrowing of the half-height bandwidth
`to 108 nm (Fig. 2B), a profile consistent with that of culture-
`derived MTT formazan.
`As shown in Fig. 2C the presence of 0.5 to 5.0% (v/v) serum
`in DMSO substantially enhances
`(2.3x maximum)
`the molar
`extinction coefficient of MTT formazan (Ey* = 15,400 versus
`£"553= 36,300 M~' cm"1)- Serum concentrations
`of 0.5 to 5.0%
`
`in 150 ß\DMSO (volume of solvent employed in final step of
`MTA) are equivalent
`to 31.5-315 pg protein/culture well (since
`the total protein concentration
`of the fetal bovine serum lot
`was 42 mg/ml). The presence of 10 ^1 (or somewhat more)
`culture medium (containing 10% FBS) which remains following
`the aspiration
`step coupled with protein adsorbed to culture
`well surfaces and cell monolayers provides an adequate amount
`of protein (more than 42 /ug) for formazan complex formation
`and nearly maximum extinction. DMSO rapidly solubilizes
`serum as well as formazan, whereas
`isopropanol,
`propanol,
`hexane, and dimethylformamide
`are incapable of solubili/in^
`serum at concentrations
`exceeding 0.0625% (data not shown).
`While serum is highly soluble in propylene glycol (>10% v/v is
`achievable),
`this solvent does not adequately mobilize cell-
`generated MTT formazan. The combined solubility of serum
`and formazan in DMSO unlike other organic solvents appears
`to be responsible for the improved extraction and detection of
`MTT formazan generated within cultured cell systems.
`The effect of serum upon formazan extinction is not unique
`to MTT formazan. DMSO and serum over the same range of
`serum concentrations
`also enhance extinction and narrow the
`absorption bandwidth of INT formazan. While the molar ex
`tinction coefficients of INT formazan in DMSO and DMSO/
`serum exceeds that of MTT formazan (Table 2), the rate of
`formazan generation by cells is more rapid for MTT than for
`INT.
`In addition, MTT formazan
`(blue violet)
`is easier
`to
`visualize than INT formazan
`(red)
`in cell culture by light
`microscopy.
`stable
`in DMSO exhibits
`reagent
`While MTT formazan
`for several days, the absor
`Spectrophotometric
`characteristics
`bance of MTT formazan derived from cell culture (under con
`ditions described for MTA above) begins to change within
`several hours and is affected by DMSO grade and stock DMSO
`storage conditions. Use of Spectrophotometric grades of DMSO
`provide stable "background"
`absorbance
`levels for up to 2 h
`following solvent application, whereas use of nonspectropho-
`tometric DMSO preparations or DMSO preexposed to air are
`accompanied by ever-increasing levels of "background"
`absor
`bance within 15 min of solvent application.
`Microculture Growth Evaluations.
`/// vitro growth evaluations
`on candidate cell lines were performed as follows. For each cell
`line a range of inoculation densities (40-20,000 cells/well) and
`culture durations (usually 1, 2,4, 8, and 11 days) were evaluated
`(e.g., Fig. 3, A and B). From such data it was possible to
`determine which inoculation densities give rise to a detectable
`and linear
`range of absorbance
`readings
`for a given culture
`duration.
`the absorbance arising from MTT forma
`To assess whether
`zan in adherent cell line cultures reflects total cell mass/well,
`replicate culture plates for each of four cell lines (A549, NCI-
`H460, NCI-H322,
`and NCI-H23) were subjected to the MTA
`
`591
`
`

`

`MICROCULTURE TETRAZOLIUM ASSAY
`
`Table 1 Established cell lines employed for initial
`
`in vitro growth evaluations and MTA development
`
`inoculation
`
`
`medium*51733171717171717171731717317171717/1917/1917/1917/1917/1917/1917/192424242317171614171551611115282814171726271722111717MTArange"78-1,250312-2,500156-5,000156-2,500156-2,500312-2,500156-5,00039-5,000312-5,000156-5,00078-5,000312-2,50039-1,250156-5,000156-5,00078-1,250312-5,00039-15678-1,250156-5,0001,250-10,000312-5,0001,250-10,000156-5,00039-2,500312-5,00039-2,500156-5,00078-1,250156-5,000312-5,00039-625156-2,50039-62539-62539-312156-5,000156-10,00039-625156-10,00039-1,25078-2,500312-5,00078-5,00039-2,50039-2,500156-1,250312-5,00039-1,25039-2,50078-5,000625-5,000625-5,000density
`
`Histology
`cell
`lineNon-small
`
`cell lungcancerAdenocarcinomaA549A549/ASC-1Calu-3Calu-6EKVXNCI-H23NCI-H324NCI-H522Adenosquamous
`
`(Source")National
`
`Cancer Institute(ATCC)National
`
`
`Cancer Institute(NCI-TB)Memorial
`
`Sloan-Kettering Cancer Center(ATCQMemorial
`
`Sloan-Kettering Cancer Center(ATCC)Norsk
`
`Hydro's Institute, Norway (O.Fodstad)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`
`Cancer Institute (A. F.Gazdar)National
`
`carcinomaNCI-HI
`25NCI-H647Squamous
`
`
`
`cellcarcinomaNCI-H520NCI-H226SK-MES-1Bronchiole-alveolar
`Cancer Institute (A. F.Gazdar)National
`
`
`Cancer Institute (A. F.Gazdar)Memorial
`
`Sloan-Kettering Cancer Center(ATCC)National
`
`carcinomaNCI-H322NCI-H3S8Large
`
`
`
`cellcarcinomaA427AHSMNCI-H460Mucoepidermoid
`
`carcinomaNCI-H292SCLC*"Classic"NCI-H69NCI-HI
`
`28NCI-H146NCI-HI
`
`87NCI-H249"Variant"NCI-H82NCI-H524"Adherent"DMS
`
`114DMS
`187DMS
`273SHP77Colon
`
`CancerCOLO
`205DLD-1HCC
`
`2998HCT116HT-29LoVoLS
`
`174TMHC
`1544SW620SW
`
`1116WiDrRenal
`
`cancerA498A704Caki-1SN12CSNI2
`
`KlUO-31Breast
`
`Cancer Institute (A. F.Gazdar)National
`
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute(ATCC)Norsk
`
`
`Hydro's Institute, Norway (O.Fodstad)National
`
`Cancer Institute (A. F.Gazdar)National
`
`
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`Cancer Institute (A. F.Gazdar)National
`
`
`Cancer Institute (A. F.Gazdar)Dartmouth
`
`Medical School (O. S.Pettengill)Dartmouth
`
`
`Medical School (O. S.Pettengill)Dartmouth
`
`Medical School (O. S.Pettengill)University
`
`of Pittsburgh (E. R.Fisher)Denver
`
`Medical Hospital(ATCC)Brown
`
`
`University(ATCC)M.
`
`D. Anderson Hospital & TumorInstitute(I.
`
`J.Fidler)Baylor
`College(ATCC)Memorial
`
`
`Sloan-Kettering Cancer Center(NCI-TB)M.
`
`D. Anderson Hospital & TumorInstitute(ATCQNorthwestern
`
`(ATCC)M.
`University Hospital
`
`D. Anderson Hospital & TumorInstitute(I.
`
`J.Fidler)Scott
`
`White Clinic(ATCC)Scott
`
`White Clinic(ATCC)Bureau
`of Biologies(ATCC)National
`
`
`Cancer Institute(ATCC)National
`
`
`Cancer Institute(ATCC)Memorial
`Sloan-Kettering Cancer Center
`(MSK)M.
`
`D. Anderson Hospital & TumorInstitute(I.
`
`J.Fidler)M.
`
`D. Anderson Hospital & TumorInstitute(I.
`
`J.Fidler)National
`Cancer Institute (W. M.Linchan)Naval
`
`
`cancerHS
`Biosciences Laboratory(ATCC)Michigan
`
`578TMCF7
`Cancer Foundation (K.Cowan)National
`
`WTMCF7
`Cancer Institute (K.Cowan)M.
`
`ADRMDA-MB-231ZR-75-1ZR-75-30Reference18—1919—20,21222221,22—21-2321,231921,2421-2418—21,222520,2220,2220,222222222226,2726,272728,293031—32193334353636371818193838—3940414243,4443,44Institution
`
`D. Anderson Hospital & TumorInstitute(ATCC)National
`
`Cancer Institute(ATCC)National
`
`Cancer Institute (ATCC)Culture
`
`592
`
`

`

`Histology
`inoculation
`(Source")Naval
`densityrange'625-5,00039-625312-5,000156-5,000625-5,000312-5,000312-2,50078-5,000312-5,000
`
`
`celllineMelanomaHs294T(A101D)LOXMalme-3MRPMI-7951SK-MEL-1SK-MEL-2SK-MEL-5SK-MEL-28SK-MEL-31Reference45—19464719484848Institution
`medium251712333334MTA
`
`MICROCULTURE TETRAZOLIUM ASSAY
`
`Table 1—Continued
`
`Biosciences Lab(ATCC)Norsk
`
`Hydro's Institute, Norway (O.Fodstad)Memorial
`
`
`Sloan-Kettering Cancer Center(ATCC)Roswell
`
`Park Memorial(ATCC)Memorial
`Institute
`
`Sloan-Kettering Cancer Center(ATCC)Memorial
`
`Sloan-Kettering Cancer Center(ATCC)Memorial
`
`Sloan-Kettering Cancer Center(ATCC)Memorial
`
`Sloan-Kettering Cancer Center(ATCC)Memorial
`Sloan-Kettering Cancer Center (ATCC)Culture
`
`50
`50
`19
`49
`50, 51
`50, 51
`50, 51
`
`19
`
`52, 53
`54
`55,56
`
`57
`58
`
`59,60
`59,60
`61
`62
`63
`64
`64
`65
`
`66,67
`66,67
`66,67
`68,69
`68,69
`68,69
`
`70
`71
`
`18
`18
`46
`72
`73
`
`National Cancer Institute (T. C. Hamilton)
`National Cancer Institute (T. C. Hamilton)
`Memorial Sloan-Kettering Cancer Center (ATCC)
`Institute Gustave Roussy, France (J. Benard)
`National Cancer Institute (T. C. Hamilton)
`National Cancer Institute (T. C. Hamilton)
`National Cancer Institute (T. C. Hamilton)
`National Cancer Institute (T. C. Hamilton)
`Memorial Sloan-Kettering Cancer Center (ATCC)
`
`Duke University (ATCC)
`Roswell Park Memorial
`Institute (MSK)
`Pasadena Center for Medical Research
`(M. E. Kaighn)
`National Cancer Institute-FCRF (M. E. Kaighn)
`University of Michigan (H. B. Grossman)
`University of Minnesota (MSK)
`
`Children's Cancer Research Foundation (ATCC)
`Children's Cancer Research Foundation (ATCC)
`National Cancer Institute (NCI-TB)
`University of Tennessee (ATCC)
`Roswell Park Memorial
`Institute (ATCC)
`Southern Research Institute (NCI-TB)
`Southern Research Institute (NCI-TB)
`Roswell Park Memorial
`Institute (ATCC)
`
`University of California (M. L. Rosenblum)
`University of California (M. L. Rosenblum)
`University of California (M. L. Rosenblum)
`NINCDS (P. L. Kornblith)
`NINCDS (P. L. Kornblith)
`NINCDS (P. L. Kornblith)
`NINCDS (P. L. Kornblith)
`Children's Hospital, Los Angeles (NCI-TB)
`University of Uppsala, Sweden (NCI-TB)
`
`National Cancer Institute (ATCC)
`National Cancer Institute (ATCC)
`Naval Biosciences Lab (ATCC)
`University of Southern California (ATCC)
`National Cancer Institute (J. S. Rhim)
`
`Ovarian cancer
`A2780
`A2780 CP70
`CAOV-3
`IGROV-1
`OVCAR 3
`OVCAR 4
`OVCAR 5
`OVCAR 8
`SK-OV-3
`
`Prostate cancer
`DU-145
`LNCaP
`PC-3
`
`PC-3M
`UMSCP-1
`1013 L
`
`Leukemia
`CCRF-CEM
`CCRF-SB
`HL-60
`K-562
`Molt-4
`P388
`P388/ADR-Resist
`RPMI 8336
`
`Central nervous system cancer
`SF126
`SF295
`SF539
`SNB19
`SNB44
`SNB56
`SNB75
`TE671
`U251
`
`Sarcoma
`A 204
`A673
`HS 913T
`HT1080
`Te85
`
`Head and neck squamous cancer
`UM-SCC-14 B,C
`UM-SCC-21 A
`UM-SCC-22 B
`
`74, 75
`74,75
`74, 75
`
`University of Michigan (T. E. Carey)
`University of Michigan (T. E. Carey)
`University of Michigan (T. E. Carey)
`
`FibroblastsCCD-19LUIMR-90Mar-BelMCR-546764677American
`Type CultureCollectionInstitute
`
`
`for Medical Research(ATCC)American
`
`Type CultureCollectionNational
`
`Institute for Medical Research,UK(ATCC)308301312-5,000312-5,000312-5,000625-5,000
`
`17
`17
`25
`17
`17
`17
`17
`17
`13
`
`3
`20
`31
`
`31
`9
`18
`
`6
`6
`21
`17
`17
`17
`17
`17
`
`2
`2
`2
`29
`29
`29
`29
`25
`30
`
`14
`5
`25
`5
`10
`
`9
`9
`9
`
`39-2,500
`78-2,500
`625-5,000
`78-625
`156-5,000
`312-5,000
`625-5,000
`78-5,000
`625-5,000
`
`78-2,500
`39-1,250
`78-5,000
`
`78-1,250
`78-1,250
`1,250-5,000
`
`1,250-10,000
`625-10,000
`78-2,500
`39-156
`312-5,000
`78-1,250 (4 day)
`10-312 (4 day)
`156-5,000
`
`78-1,250
`39-1,250
`156-10,000
`39-2,500
`156-5,000
`39-2,500
`78-5,000
`156-5,000
`39-5,000
`
`78-5,000
`156-2,500
`156-5,000
`78-2,500
`78-5,000
`
`156-2,500
`78-5,000
`39-625
`
`investigator were as follows: ATCC, American Type Culture Collection; MSK, Memorial Sloan-Kettering Cancer Center
`" Cell line sources if other than original
`(Walker Laboratory); and NCI-TB, NCI-Division of Cancer Treatment Tumor Bank.
`'Culture medium formulations
`recommended by source were as follows: 1 = BME, 10% FBS, Hank's BSS; 2 = EMEM, 10% FBS, NEAA, L-glulamine,
`gentamicin; 3 - EMEM, 10% FBS, NEAA, pyruvate; 4 = EMEM, 15% FBS, NEAA, pyruvate; 5 = EMEM, 10% FBS, NEAA, Earle's BSS; 6 = EMEM, 10% FBS
`(modified for suspension); 7 = EMEM, 10% FBS, NEAA, pyruvate, MEM vitamins; 8 = EMEM, 10% FBS, NEAA; 9 = EMEM, 15% FBS, P/S; 10 = EMEM,
`10% FBS, P/S; 11 = L 15, 10% FBS; 12 = L 15, 15% FBS; 13 - McCoy's 5A, 15% FBS; 14 = McCoy's 5A, 10% FBS; 15 = Ham's F12, 20% FBS; 16 = Ham's
`F12, 15% FBS, EOF,
`transfemn,
`insulin; 17 = RPMI 1640, 10% FBS; 18 = RPMI 1640, 15% FBS, P/S; 19 = RPMI 1640, hydrocortisone,
`insulin,
`transferrin,
`EGF, selenium; 20 = RPMI 1640, 10% FBS, 2x L-glutamine; 21 = RPMI 1640, 20% FBS; 22 = RPMI 1640, 10% FBS, doxorubicin HC1 (5 MM);23 = RPMI 1640,
`9% FBS; 24 = Waymouth's
`752/1, 10% FBS, P/S; 25 = DMEM, 10% FBS, 4.5 g/liter glucose; 26 = DMEM, 10% FBS, 4.5 g/liter glucose, HEPES [4-(2-
`hydroxyethylH-piperazineethanesulfonic
`acid], P/S/fungizone/gentamicin;
`27 = DMEM,
`10% FBS, 4.5 g/liter glucose,