prenatal/cytogenetics product catalog - irvine · pdf file cytogenetics and diagnostic...

Prenatal/Cytogenetics Product Catalog

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Cytogenetics and Diagnostic ProductsHeadline for TOC

Irvine Scientific Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Quality Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4Ordering and Shipping Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8

cytOgenetIcS and DIAgnOStIc PRODUctS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 clinical Laboratory Products for Fetal Lung Maturity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 chang Medium® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11-14 cell culture Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 cell culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16-18 Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19-20 Reagents and Media components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-22

tecHnIcAL InFORMAtIOn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 cryopresevation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25-30

InDeX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31-32

ContentsIrvine Scientific

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Irvine Scientific ProfileIntroduction

IntRODUctIOn tO IRVIne ScIentIFIcIrvine Scientific was established in May 1970 to produce serum products for the cell culture market. From those beginnings, Irvine Scientific emerged and grew to become a respected innovator of cell culture products. Over the years, Irvine Scientific has evolved its products, services, and operations to become multi-disciplined, international in scope and subject matter experts in the area of cell culture media.

In 1987, Japan Energy (JE, a member of Nippon Mining Holdings), acquired Irvine Scientific. With the steady backing of JE (now a member of JX Holdings) Irvine Scientific became a leader in the expanding IVF and biopharmaceutical fields. JE has provided the strength to acquire products like Hana Biologicals, expand operations in California, Europe, and most recently build additional manufacturing capabilities in Japan. The second manufacturing site provides complete redundancy of commercial phase products, which reduces risks of product supply interruption and expertly positions us to serve the emerging markets in Asia.

Today, our diversified products and services benefit both life science and medical customers. Irvine Scientific is proud of its past as an innovator of products that positively touch so many lives. Whether it is developing the highest quality embryo culture media to assist IVF procedures, to supplying high quality media for diagnostics and research, to customizing media for biopharmaceutical production, providing the highest customer satisfaction is our primary goal. We plan to build on this proud history and continue innovating to meet our customer needs

QUALIty POLIcyIrvine Scientific strives to be the top performer for our clients across all business units and build relationships of trust with our customers.

We establish, review and communicate our Quality Objectives throughout our organization, to assure understanding, continuing suitability and to maintain the effectiveness of the Quality Management Systems.

Our quality management system is committed to comply with the requirements applicable to the markets we serve.

MISSIOnIrvine Scientific’s mission is to set the standard for EXCELLENCE through:

• QualityProductsfortheCellCulture,Diagnostics,and Biopharmaceutical Markets• QualityServicewithrespect,courtesyandloyaltytoourcustomers• QualityPeoplededicatedtocompletecustomersatisfaction• Customersatisfactionisnotourgoal…itisourpolicy!

IRVIne ScIentIFIc cORPORAte cULtURe MISSIOnIn keeping with our commitment to quality, we expect every team member to demonstrate integrity and accountability to one’s self, our colleagues and the business of Irvine Scientific

We are committed to mutual respect, honesty and leadership by example.Irvine Scientific requires a strong work ethic and open communication at every level.

Integrity:Doing theright thing, in the rightway for the right reasonswithout compromise.

Accountability: Owning your responsibilities, actions, and the results of your actions, whether good or bad, as individuals and collectively as team members.

Mutual Respect: Showing consideration for one another and appreciation of each other’s contributions to the company.

Honesty: Being truthful.

Leadership by Example: Ensuring that the expectations you have of others is reflected in your own actions.

Work Ethic: Being self-motivated to commit one ‘s full effort each workday.Workasthoughyouownpartofthecompany!

Open Communication: Speaking candidly, expressing true opinions and listening effectively throughout all levels of the organization.

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QUALIty SySteMSWe take pride in the fact that our quality systems are second to none by offering:

•Outstandingvalidationsprograms

•Strongtechnicalsupportforcustomers

•Documentationandtraceabilityforallmanufacturedlots

•Unsurpassedregulatorystrength

•Consistentlyexpedientresponsetocustomers

•Excellenttrackrecordforauditsandinspections

•Corporateculturefocusonvaluesandcustomers

ISO 13485 ceRtIFIcAtIOnIrvine Scientific has received Certification to the ISO 13485 International Quality Standard. The Certification was achieved after completing an assessment of the quality management system against the comprehensive requirements of the world-class standard to the manufacture of medical devices. This certification illustrates the commitment of Irvine Scientific to providing quality products to the industrial, research, and laboratory biomedical communities worldwide.

FDA cLeARAnceWhererequired,ourproductshavebeenclearedbytheFDAincomplianceto510Ks.DrugMasterFiles(DMF)forourkeyproductshavebeenfiledwiththeFDAtofurtherassisttheregulatoryneedsofourcustomers.

ce MARKIngWhere required our products meet European requirements for CE Marking.

MAnUFActURIngLiquid Media. Approved filtration and filling protocols include testing for both filter integrity and product quality. Media is filtered through 0.1 micron filters to achieve a Sterility Assurance Level (SAL) of 10-3. Aseptic fillingandcappingareperformedwithinaClass100environment.Productis labeled and immediately sent to storage. Finished product testing is performed on media in accordance with written Standard Operating ProceduresandFinishedProductSpecificationsasarequirementfor finalrelease.

Powdered Cell Culture Media and Balanced Salts are prepared in accordance with the original formula for liquid when feasible. Anhydrous salts are substituted on an equimolar basis. Powder is packed in screwcapped containers and should be stored dry according to product labeling. Small aliquots may be removed for use if appropriate concern for moisture is exercised. Powderedmedia is checked for particle size uniformity andcomplete solubility. Irvine Scientific uses procedures to achieve thorough distribution of trace compounds in powdered media.

Chemicals used in Irvine Scientific media products conform to at least ACS, USP, NF and other International Pharmacopeia standards whereavailable. Certificates are obtained from vendors. Samples of components are tested at a minimal for endotoxin, identity (by Fourier Transform Infrared Spectroscopy), efficacy and toxicity as appropriate.

Human Source Raw Materials used in the manufacturing of Irvine Scientific reproductive media are therapeutic grade. All human source material has been tested at the donor level with FDA licensed kits,and found to be nonreactive for the antibodies to Hepatitis B Surface Antigen (HBsAg), antibodies to Hepatitis C (HCV) and antibodies to Human Immunodeficiency Virus (HIV 1&2). Source material is also screened by questionnaire at the donor level for risk factors associated with Creutzfeldt-JakobDisease(CJD).

Water. The water system is routinely monitored for microbial levels, endotoxin, conductivity, and TOC (total organic carbon). Finished product watermeetsUSPtestrequirementsfor“WaterforInjection”.

QUALIty cOntROLSterility Testing of all media is performed in accordance with the Code of Federal Regulations (CFR) Title 21, Part 610.12 or current USP<71>. Statistical samples are filtered through 0.45 micron membranes. The membranes are incubated at 30°-35°C in Fluid Thioglycollate and at 20°-25°C in Soybean-CaseinDigestmedia andobserved regularly for 14days.

0050

Quality Systems

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Quality Systems

Endotoxin is measured using the LAL gel clot or kinetic chromogenic methodology on all Media and is performed in accordance with current USP<85>.Productscontainingproteinareheattreatedinboilingwaterfor2 minutes to denature the enzymes that interfere with the enzyme cascade reaction for endotoxin detection.

pH is measured at a 1X concentration in accordance with finished product specifications. The user is reminded that a medium in free exchange with CO2 in an incubator may establish a final equilibrated pH that is different from the initial manufactured pH. The user should always check equilibrated aliquots of the final media with a pre-calibrated pH meter rather than relying on visual estimation alone, which can be deceptive.

Osmolality testing is performed either by the freezing point or vapor pressure methodology. Testing is performed on precalibrated instrumentation using specificStandardOperatingProcedurestoensureaccuracyandconsistency.

Certificates of Analysis (CofA) are provided with each shipment, and test results are reported on a lot-specific basis.

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PRIcIng AnD teRMSIrvine Scientific reserves the right to change prices without notice. Any price reduction will automatically apply to your invoice. Invoices are due in net 30daysinU.S.dollars.Afinancechargeof1.5percentpermonth(annualpercentage rate of 18 percent) will be charged to past due accounts.

LOt ReSeRVAtIOn POLIcySamples will be provided at no charge for the purpose of testing a particular lot. Reserved lots will be held for three weeks from the date the sample is shipped. Upon completion of your testing, a purchase order for the amount reserved is required to secure product. The reserve will be automatically cancelled at the end of the three weeks unless otherwise notified.

cReDIt cARD ORDeRSFor your convenience Irvine Scientific accepts VISA®, MasterCard® and American Express® as payment for your order. Please provideyour credit card account number and expiration date when you place your order.

PeRISHABLe PRODUct PAcKAgIngPerishableproductswillbepackedasappropriate in insulatedcartons forsafe arrival. A packaging charge will be added as a separate item on our invoice when products require dry ice or cold packaging. We will not accept responsibility for shipments packaged contrary to our procedures at the customer’s request.

United States HeadquartersPhone 1(800)437-5706 1(949)261-7800 Fax 1(949)261-6522E-mail [email protected] www.irvinesci.com

European HeadquartersPhone 3531281-99-20Fax 353 1 281-99-28E-mail [email protected]

General Information Domestic and International Ordering

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General Information Shipping

SHIPPIngItemsorderedareshippedwithin48hoursofreceiptoftheorder.Partialshipments may be made when complete orders cannot be shipped. If partial shipments are not acceptable, indication should be made at the time the order is placed.

Orders are shipped via best way unless otherwise specified. Irvine Scientific routinely ships via FedEx®.

Shipments are FOB origin, freight prepaid. Title to the items sold passes to the purchaser upon delivery of the items to the carrier.

SHORtAgeS AnD DAMAgeSIf a shortage occurs in any shipment received from Irvine Scientific, please notify our Customer Service Department within three business days fora credit or replacement product. Otherwise, the order will be considered complete.

Irvine Scientific provides assistance with filing claims for loss or damaged product.Pleasekeepallpackingmaterialsandcontainersforinspectionbythe carrier.

cAnceLLAtIOnSCancellation of custom prepared products will not be accepted after manufacturing has begun.

RetURnSA Return Goods Authorization number and shipping instructions must be obtained fromourCustomer ServiceDepartment prior to returning anyproduct. All product must be returned in good condition and is subject to a handling and restocking charge of 20 percent plus freight. No product may be returned after 30 days.

Where spoilage occurs in transit, please advise Customer Service and request instructions immediately upon receipt of the shipment. Credit will not be issued on any items that are returned without prior authorization.

cOnDItIOnSUnless otherwise indicated, the products listed herein are for purposesdescribed only. They are not to be used for administration to humans or for drug purposes. Nothing disclosed herein is to be construed as a recommendation to use products in violation of any patents. The information presented is believed to be accurate. However, said information and products are offered without warranty or guarantee since the ultimate conditions of use and the variability of the materials treated are beyond our control. We cannot be responsible for patent infringements or other violations that may occur with the use of these products.

The information presented in this catalog concerning product specifications, descriptions, price, returns, and conditions is effective January 2008 and supersedes all previous publications.

IntenDeD USeUnless otherwise indicated, the products listed herein are for assistedreproductive technology or research use only. Not for drug use. Consult labeling.

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REDUCE • RE-USE • RECYCLEIrvine Scientific is concerned with the environmental impact of our packaging materials and we encourage our customers to dispose of these materials responsibly.Effectivere-useandrecyclingofthesecomponentsdependsonyou.Pleaseusethefollowingguidelinestohelpusprotectourenvironmentfor future generations:

Packaging Material Re-use Recycle

Glass-Type 1 Borosilicate Not recommended for re-use Not currently recyclable

Plastic-PETG Notrecommendedforre-use Disinfectedbottlescanberecycledwith Plastic-PET consumerPET(sodabottles) Incineration (results in CO2 + H20)

Plastic-HDPE Notrecommendedforre-use Disinfectedbottlescanberecycledwith consumerHDPE

Bubblewrap-PE Re-useaspackagingmaterial ContactRecycleHotLine800/944-8448 for the nearest collection site

PolystyreneFoam(EPS) Delivertocommercialpackaging ContactRecycleHotLine800/944-8448 CFC Free outlets (i.e. Mail Boxes, Etc.) for re-use for the nearest collection site


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Irvine Scientific’s Global Presence

ARGENTINAAUSTRALIAAUSTRIABAHRAINBANGLADESHBELGIUMBOLIVIABRAZILCANADACHILECHINACOLOMBIACOSTARICACYPRUSCZECHREPUBLICDENMARKDOMINICAN REPUBLIC ECUADOR EGYPT ESTONIA FINLAND FRANCE GERMANYGREECEGUAMGUATEMALAGUYANAHONDURASHONGKONGHUNGARYICELANDINDIAINDONESIAIRELANDISRAELITALYJAMAICAJAPANJORDANKOREAKUWAITLATVIALEBANONLIBYALITHUANIALUXEMBOURGMALAYSIAMEXICONETHERLANDSNEWZEALANDNIGERIANORWAYPANAMAPARAGUAYPERUPHILIPPINESPOLANDPORTUGAL PUERTORICO RUSSIA SAUDIARABIA SINGAPORE SOUTHAFRICA SPAINSWEDENSWITZERLANDTAIWANTHAILANDTURKEYU.A.E.UKRAINEUNITEDKINGDOMUNITEDSTATESURUGUAYVENEZUELA

For the local distributor of Irvine Scientific products in your country,pleasecontactourUSACustomerServiceDepartmentorvisitourwebsite:www.irvinesci.comPhone:001-949-261-7800Fax:001-949-261-6522

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clinical Laboratory Products for Fetal Lung Maturity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

chang Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

cell culture Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

cell culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Reagents and Media components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

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Cytogenetics and Diagnostic ProductsContents

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Cytogenetics and Diagnostic ProductsClinical Laboratory Products for Fetal Lung Maturity

Catalog Number Description Size

91030-006 AMINOSTAT-FLM®-PG 6patient/kit Storage: Kit 2° to 8°C For in vitro diagnostic use

AmnioStat-FLM®-PGisarapid,qualitative,slideagglutinationassayforphosphatidylglycerol(PG)inamnioticfluid.AnalysisofamnioticfluidforPGiswidelyacceptedasareliablemethodfordeterminingfetallungmaturity.ThepresenceofPGindicatesfetallungmaturityandfreedomfromdevelopingRespiratoryDistressSyndrome(RDS)upondelivery.Forthisrapid,easy-to-useassay,25µLofamnioticfluidfromtransabdominalamniocentesisorvaginalpoolsmaybeused.Unlikeothertestsforfetallungmaturity,resultsarenotaffectedbythepresenceofmoderatebloodandmeconiumcontamination,orvaginal secretions. Results are available in as little as 15 minutes providing around the clock STAT testing capability.

EachAmnioStat-FLM-PGkitcomeswith3levelsofcontrols,regents,andslides.

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Chang Medium®

CHANG AMNIO™ is a complete, ready-to-use medium with an antibiotic developed for the culture of human amniotic fluid cells, chorionic villi sampling, and tissues for use in karyotyping and other antenatal testing.

CHANG MEDIUM® C, In Situ, and D are evaluated on both primary amniocytes and early passage amniocytes. Early passage amniocytes supply the most consistent and reproducible results while primary amniocytes more accurately reflect the products’ use in the clinical cytogenetics laboratory.

CHANG MARROW™ is a complete, ready-to-use medium optimized to support bone marrow cultures for karyotyping and other genetic testing of various hematological disorders. The product is tested on clinical bone marrow specimens and evaluated for suitable chromosome morphology to detect acquired clonal abnormalities in hematologic dysplasia.

CHANGMEDIUM®BMCisevaluatedfor itsabilitytosupportmitoticactivityofbonemarrowcells.Theproductistestedonclinicalbonemarrowspecimens and evaluated for suitable chromosome morphology to detect acquired clonal abnormalities in hematologic dysplasia.

CHANGMEDIUM®MFismitogen-free,designedforhemopoieticcells.BecauseCHANGMEDIUM® MF can be used for both unstimulated peripheral blood and bone marrow cultures, it is tested on both peripheral blood and bone marrow specimens.

Catalog Primary Early Passage Bone Peripheral Number Description Amniocytes Amniocytes Marrow Blood

99473 CHANG AMNIO™ • •C100-C106 CHANGMEDIUM® C • •

C101-C108

T101-019

T101-059

T104 CHANGMEDIUM® In Situ • •

T105 CHANGMEDIUM®D • •

91031 CHANG MARROW™ •91004 CHANGMEDIUM® BMC •

91005 CHANGMEDIUM® MF • •

Cytogenetics and Diagnostic Products

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Cytogenetics and Diagnostic ProductsChang Medium®


99473 CHANG AMNIO™ 100 mL Storage: -10° C 500 mL For in vitro diagnostic use

C100 CHANGMEDIUM® BASAL 90 mL Storage: Basal 2° to 8° C For in vitro diagnostic use

C101 CHANGMEDIUM® BASAL 450 mL Storage: Basal 2° to 8° C For in vitro diagnostic use

C106 CHANGMEDIUM®C,FROZENSUPPLEMENT 14mL T Storage: Below -10° C For in vitro diagnostic use

C108 CHANGMEDIUM®C,FROZENSUPPLEMENT 70mL T Storage: Below -10° C For in vitro diagnostic use

99419 CHANGMEDIUMC,LYOPHILIZEDKIT 100mL w/GentamicinandAlanyl-Glutamine 500mL

T101-019 CHANGMEDIUM®C,LYOPHILIZEDKIT 100mL Storage: 2° to 8° C For in vitro diagnostic use

T101-059 CHANGMEDIUM®C,LYOPHILIZEDKIT 500mL Storage: 2° to 8° C For in vitro diagnostic use

PRENATAL

T shipped with dry ice

cHAng AMnIO™CHANG AMNIO™ is a complete, ready-to-use medium with an antibiotic developed for the culture of human amniotic fluid cells, chorionic villi sampling for use in karyotyping and other antenatal testing. With an improved blend of basal media and sera, Irvine Scientific’s Chang Amino™ is shown to yield significantly higher growth than other commercially available media.

cHAng MeDIUM® cCHANGMEDIUM® C was developed for the primary culture of human amniotic fluid cells for use in karyotyping and other antenatal genetic testing. This formula has been optimized for both open and closed systems.

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Cytogenetics and Diagnostic ProductsChang Medium®

PRENATAL


T104 CHANGMEDIUM® In Situ 100 mL T packaged in single bottles 500 mL ready-to-use with no mixing required Storage: Below -10°C For in vitro diagnostic use


T105 CHANGMEDIUM®D 100mL T packaged in single bottles 500 mL ready-to-use with no mixing required Storage: Below -10°C For in vitro diagnostic use 99404 CHANGMEDIUM®DWITHGENTAMICIN 100mL T packaged in single bottles 500 mL ready-to-use with no mixing required Storage: Below -10°C For in vitro diagnostic use


cHAng MeDIUM® In Situ CHANGMEDIUM® In Situ was developed for the primary culture of human amniotic fluid cells for use in karyotyping and other antenatal genetic testing. This formula has been optimized for in situ methodologies.

cHAng MeDIUM® DCHANGMEDIUM®Dwasdevelopedfortheprimarycultureofhumanamnioticfluidcellsforuseinkaryotypingandotherantenatalgenetictesting. This formula has been optimized for both flask and in situ methodologies.

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Cytogenetics and Diagnostic ProductsChang Medium® POSTNATAL


91031 CHANG MARROW™ 100 mL T packaged in single bottles 500 mL ready-to-use with no mixing required Storage: Below -10° C For in vitro diagnostic use


91004 CHANGMEDIUM® BMC 100 mLT packaged in single bottles 500 mL ready-to-use with no mixing required Storage: Below -10° C For in vitro diagnostic use


cHAng MARROW™CHANG MARROW™ is a complete, ready-to-use medium optimized to support bone marrow cultures for karyotyping and other genetic testing of various hematological disorders. After an extensive optimization process to create a superior, innovative blend of growth factors, Irvine Scientific’s brand new medium Chang Marrow™ has been shown to out-perform MarrowMAX™ in clinical studies. Each lot has been performance tested on Clinical Bone Marrow Cultures at an independent Clinical Cytogenetics Laboratory. The product is tested on clinical bone marrow specimens and evaluated for suitable chromosome morphology to detect acquired clonal abnormalities in hematologic dysplasia.

BOne MARROW cULtURe MeDIA

BOne MARROW, PeRIPHeRAL BLOOD, AnD OtHeR t-ceLL LyMPHOcyte cULtURe MeDIA

cHAng MeDIUM® BMcDevelopedfortheprimarycultureofClinicalHumanBoneMarrowCulturesforuseinkaryotypingandothergenetictestingusingexistingprotocolsbasedonRPMIMedium1640,10%GCT-CMandgentamicin.EachlothasbeenperformancetestedonClinicalBoneMarrowCulturesatanindependentClinical Cytogenetics Laboratory.

cHAng MeDIUM® MF, MItOgen-FRee FOR HeMOPOIetIc ceLL cULtUReDeveloped for the primary culture of Clinical Human BoneMarrow Cultures, Peripheral Bloods and other T-Cell Lymphocyte cultures for use inkaryotypingandothergenetictestingusingexistingprotocolsbasedonRPMIMedium1640,20%FBSandgentamicin.EachlothasbeenperformancetestedonClinicalBoneMarrowCulturesandPeripheralBloodCulturesatanindependentClinicalCytogeneticsLaboratory.


91005 CHANGMEDIUM® MF 100 mL T packaged in single bottles 500 mL Available by Standing Order Only ready-to-use with no mixing required Storage: Below -10° C For in vitro diagnostic use

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Cytogenetics and Diagnostic ProductsCell Culture Sera


2014 FORMULAFEDBOVINECALFSERUM 100mLT iron supplemented 500 mL lessthan16weeks Storage: Below -10°C

3000A FETALBOVINESERUM 50mLT cell culture grade 100 mL includes extensive documentation 500 mL Storage: Below -10°C

ceLL cULtURe SeRACell culture sera are processed through three 0.1 micron filters. Sera are tested for growth-promoting characteristics and absence of toxicity in appropriate cell systems. All sterile filtered sera are mycoplasma, endotoxin, and virus tested. Fetal Bovine Serum is screened for folic acid.


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Cytogenetics and Diagnostic ProductsCell Culture Media


9024 DMEHIGHGLUCOSEWITHOUTL-GLUTAMINE 500mL with4500mg/Lglucose 1000mL with3700mg/Lsodiumbicarbonate without sodium pyruvate withoutL-glutamine,add20mL/L(200mM) Storage: 2° to 8°C 9031 DMEHIGHGLUCOSE 500mL with L-Glutamine 1000 mL with 4500 mg with3700mg/Lsodiumbicarbonate without sodium pyruvate Storage: 2° to 8°C

9041 DEFICIENTDMEHIGHGLUCOSE 500mL with4500mg/Lglucose with3700mg/Lsodiumbicarbonate without phenol red withoutL-glutamine,add20mL/L(200mM) with sodium pyruvate Storage: 2° to 8°C

DMe DULBeccO’S MODIFIcAtIOn OF eAgLe’S BASAL MeDIUM (BMe)Withafour-foldincreaseinaminoacidsandvitamins,alongwithadditionalnutrients,DMEprovidessupportforgrowthofawidevarietyof cell lines. Originally used for embryonic mouse and virally infected hamster cells.

DeFIcIent DMe HIgH gLUcOSeOriginallyforusewithembroyonicmouseandvirallyinfectedhamstercells,DME’sfourfoldincreaseofaminoacids,vitamins,andnutrientsprovidesupport for a wide variety of cell lines.


9032 ISCOVE’SMODIFIEDDULBECCO’SMEDIUM 500mL with25mMHEPESbuffer 1000mL with3024mg/Lsodiumbicarbonate without alpha-thioglycerol withoutL-glutamine,add20mL/L(200mM) Storage: 2° to 8°C

IMDM IScOVe’S MODIFIeD DULBeccO’S MeDIUMAmodificationofDulbecco’sBMEusinghighglucoseandpyruvate,additionalaminoacids,25mMHEPES,seleniumandothercomponents.Designedforserum-freegrowthofprimaryhemopoieticcellsinculture.Additionallysupportsthegrowthoflymphocytes,hybridomasandavarietyofhybrid cells.

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HAM’S nUtRIent MIXtUReOriginallydevelopedforcloningChineseHamsterOvaryandlungcells.Useful,withserumsupplements,foravarietyofserum-freeapplicationsincluding the growth of myeloma and hybridoma cells.

17



9056 HAM’sF-10 500mL with1200mg/Lsodiumbicarbonate withoutL-glutamine,add5mL/L(200mM) Storage: 2° to 8°C

9058 HAM’S F-12 500 mL with1176mg/Lsodiumbicarbonate withoutL-glutamine,add5mL/L(200mM) without linoleic acid Storage: 2° to 8°C

9052 HAM’SF-12/DMEHIGHGLUCOSE1:1MIXTURE 500mL with3151mg/Lglucose 1000mL 1200mg/Lsodiumbicarbonate withoutL-glutamine,add12.5mL/L(200mM) without linoleic acid Storage: 2° to 8°C

9077 HAM’sF-12K(KAIGHN’SNUTRIENTMIXTUREF-12) 500mL with2500mg/Lsodiumbicarbonate 1000mL without ascorbic acid without linoleic acid withoutL-glutamine,add10mL/L(200mM) Storage: 2° to 8°C

9195 CLICK’SMEDIUM(EHAA) 500mL with1350mg/Lsodiumbicarbonate without mercaptoethanol withoutL-glutamine,add20mL/L(200mM) Storage: 2° to 8°C

MccOy’S MeDIUM 5A (Iwakata and Grace Modification)A general medium for primary and established cell lines using increased glucose, additional vitamins, high phosphate and peptone.


9090 McCOY’SMEDIUM5A 500mL with3000mg/Lglucose with2200mg/Lsodiumbicarbonate withoutL-glutamine,add7.51mL/L(200mM) Storage: 2° to 8°C

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9160 RPMIMEDIUM1640 100mL 2000mg/Lsodiumbicarbonate 500mL withoutL-glutamine,add10.3mL/L(200mM) 1000mL Storage: 2° to 8°C

9161 RPMIMEDIUM1640WITHL-GLUTAMINE 100mL 2000mg/Lsodiumbicarbonate 500mL Storage: 2° to 8°C

9159 RPMIMEDIUM16401XHEPES* 100mL 25mMHEPESbuffer 500mL withoutL-glutamine,add10.3mL/L(200mM) *Osmolalitymodified:4587.28mg/LHEPES,acidform 1496.73mg/LHEPES,1NaSalt Storage: 2° to 8°C

9157 RPMIMEDIUM16401XHEPES*WITHL-GLUTAMINE 500mL 25mMHEPESbuffer *Osmolalitymodified:4468mg/LHEPES,acidform 1627mg/LHEPES,1NaSalt Storage: 2° to 8°C

9154 MODIFIEDRPMI1640 100mL with2000mg/Lsodiumbicarbonate without folic acid withoutL-glutamine,add10.3mL/L(200mM) Storage: 2° to 8°C

RPMI MeDIUM 1640Designedforgrowthofhumanleukemiacellsineithermonolayersorsuspensionandusefulforawidevarietyofsuspensionandmonolayerculturesandfor hybridomas.

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Cytogenetics and Diagnostic ProductsSalt Solutions

ALPHA MeM MeDIUM AModifiedEagle’sMinimumEssentialMediumsupplementedwithaminoacids,vitamins,andnucleosides.Designedforgrowthofamouse-hamsterhybridoma cell line, 8-A (Stanners et al, 1971).

MeM eAgLe’S MInIMUM eSSentIAL MeDIUMAn uncomplicated medium useful for a wide range of species and cells.

Catalog Number Description Size 9126 MEM1XEARLE’SSALTS 500mL without non-essential amino acids 1000 mL 2200mg/Lsodiumbicarbonate withoutL-glutamine,add10mL/L(200mM) Storage: 2° to 8°C

9130 MEM NEAA 1X EARLE’s SALTS 500 mL with2200mg/Lsodiumbicarbonate with non-essential amino acids withoutL-glutamine,add10mL/L(200mM) Storage: 2° to 8°C


9144 ALPHAMEMEARLE’SSALTS 100mL with deoxyribonucleosides 500 mL with ribonucleosides 2200mg/Lsodiumbicarbonate withoutL-glutamine,add10mL/L(200mM) Storage: 2° to 8°C

9142 ALPHAMEMEARLE’SSALTSWITHOUTNUCLEOSIDES 100mL without nucleosides 500 mL 2200mg/Lsodiumbicarbonate withoutL-glutamine,add10mL/L(200mM) Storage: 2° to 8°C

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Cytogenetics and Diagnostic ProductsSalt Solutions

HBSS - HAnKS’ BALAnceD SALt SOLUtIOn

Catalog Number Description Size 9220 HBSS1X-HANKS’BALANCEDSALTSOLUTION 100mL with calcium and magnesium salts 500 mL with phenol red with sodium bicarbonate Storage: 15° to 30°C

9228 HBSS1X-HANKS’BALANCEDSALTSOLUTION 100mL with phenol red 500 mL with sodium bicarbonate without calcium and magnesium salts Storage: 15° to 30°C

9230 HBSS10X-HANKS’BALANCEDSALTSOLUTION 500mL with phenol red without calcium and magnesium salts without sodium bicarbonate Storage: 15° to 30°C

PBS - DULBeccO’S PHOSPHAte BUFFeReD SALIne SOLUtIOn


9236 PBS1X-DULBECCO’SPHOSPHATEBUFFERED 500mL SALINESOLUTION 1000mL with calcium and magnesium salts without phenol red without sodium bicarbonate Storage: 15° to 30°C

9240 PBS1X-DULBECCO’SPHOSPHATEBUFFERED 100mL SALINESOLUTION 500mL without calcium and magnesium salts 1000 mL without phenol red without sodium bicarbonate Storage: 15° to 30°C

9242 PBS10X-DULBECCO’SPHOSPHATEBUFFERED 500mL SALINESOLUTION without calcium and magnesium salts without phenol red without sodium bicarbonate Storage: 15° to 30°C

9281 POTASSIUMCHLORIDESOLUTION0.075N 100mL 5.59gpotassiumchloride/literofwater 500mL Storage: 15° to 30°C

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Cytogenetics and Diagnostic ProductsReagents and Media Components


9317 L-GLUTAMINESOLUTION 100mL T 29.2mg/mL(200mM)/salinesolution Storage: below -10°C

9319 HEPESBUFFERSOLUTION1M 100mL 238.3g/Linwater Storage: 2° to 8°C

9309 WATERFORINJECTION(WFI)GRADEWATER* 1L suitable for cell culture, in vitro diagnostic 20 LC and further manufacturing use meetsUSPcriteriaforpackagedWFI Storage: 15° to 30°C 9311 COLCEMID®SOLUTION 12x10mL withSEPTUMCAPS 10mL pipette and syringe accessible 10µg/ml prepared in Hanks’ Balanced Salts Storage: 2° to 8°C

96691 PHYTOHEMAGGLUTININ-PHA(REAGENTGRADE) 5mL lyophilized powder, reconstitute with 5 mL sterile water approximately45mg/vial Storage: 2° to 8°C For in vitro diagnostic use

91006 ISGIANTCELLTUMOR-CONDITIONEDMEDIUM** 50mL T Easily grows hematopoietic cells. May be used to improve bone marrow cytogenetic analysis, isolate and recover HIV and produce and grow human hybridomas. A unique conditioned medium derived from a human tumor cell line, GCT-CM provides a winning combination ofGM-CSF,G-CSF,M-CSF,IL-1andIL-6,toensuredependable performance in a wide range of applications. Storage: Below -10°C ForResearchUse

LIQUID AntIBIOtIcS


9355 GENTAMICINSULFATESOLUTION 20mL 50mg/mLgentamicin(base)inwater recommend:1mL/Lmedium Storage: 15° to 30°C

*WFIGRADEWATERmaynotbesoldforuseinreproductiveproceduresorA.R.T.purposes.Notforparenteraluse.T shipped with dry iceColcemid® is a trademark of Ciba-Geigy Corporation**Checkforavailability

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Cytogenetics and Diagnostic ProductsReagents and Media Components

1 Trypsin1:250(PorcineParvovirusTested)1gramwilldigest250gramsofCaseinunderN.F.testforCaseindigestivepowerinpancreatinT shipped with dry ice

enZyMeS


9314 EDTA(VERSENE)SOLUTION0.526mM(1:5000) 100mL T 200mgEDTAtetrasodiumsalt phosphate buffered saline solution without calcium and magnesium salts Storage: 15 to 30°C

9336 TRYPSIN(1:250)12.5%10XSOLUTION 100mL T 25g/Ltrypsininnormalsaline dilute in a balanced salt solution without calcium and magnesium salts without phenol red Storage: Below -10°C

9340 TRYPSINEDTA1XSOLUTION 100mL T 0.5g/Ltrypsin(1:250)1 and 0.2g/LEDTAtetrasodiumsaltinnormalsaline with phenol red Storage: Below -10°C

9341 TRYPSINEDTA1XSOLUTION 100mL T 0.5g/Ltrypsin(1:250)1 and 500 mL 0.2g/LEDTAtetrasodiumsaltin hanks’ balanced salt solution without calcium and magnesium salts without sodium bicarbonate with phenol red Storage: Below -10°C

9342 TRYPSINEDTA10XSOLUTION 100mL T 5.0g/Ltrypsin(1:250)1 and 2.0g/LEDTAdisodiumdihydrateinnormalsaline without phenol red Storage: Below -10°C

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Technical InformationContents

Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

cryopresevation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

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Technical InformationAntibiotics

This table is provided as a guide in antibiotic selection. A comprehensive pharmacology text should be consulted for information on antibiotic incompatibilities and all other properties not included in this table.

Product Catalog # Inhibits1 Functional Mechanism Suggested Conc2 Stability at 37° C3 Storage

Gentamicin Sulfate Solution,

50mg/mLH₂O

9355 POS,NEG,Mycoplasma

Binds to ribosome at 30S subunit,interfering with

bacterial protein synthesis

1mL/L 5Days 15° to 30°C

1 POS=GramPositiveBacteria NEG=GramNegativeBacteria

2 ConcentrationforSolutions=mLliquid/Lmedium ConcentrationforPowdersisreportedinmilligramsofantibiotic/Lmedium;

the appropriate quantity to add per liter of medium should be adjusted by the antibiotic content of the lot of powder.

3 DatafromTCAManual

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Technical InformationCryopreservation Techniques

IntRODUctIOn cryo – relating to low temperatures preservation – to keep safe, guard, protect – maintain – to keep from decaying

A large variety of tissue cells (Loretz, et. al., 1989), microorganisms (Simione, et. al., 1977), and even embryos (Rall, et. al., 1983) have been preserved by freezing.Atverylowtemperatures(-196°C)cellularmetabolismessentiallystops, allowing living cultures to be preserved for years. The important steps in cryopreservation occurwhen the cells pass through the critical freeze/thaw equilibrium temperature range (-10°C to 40°C). Cell damage is caused by formation of ice from intracellular water at these temperatures. A good review on the mechanisms of cell freezing has been written by Mazur (1984), and is summarized here.

In freezing a cell suspension, the intracellular cytoplasm and surrounding medium are initially liquid. Due to the freeze medium salts and othercomponents, the surrounding medium does not freeze until the temperature is between -5°C and 15°C. If the cell suspension cools slowly, the cells dehydrate by exo-osmosis (ie. water flows out of the cells) into the media. This beneficial dehydration prevents ice crystals from forming inside the cell. If the cell suspension is cooled too rapidLy, the intracellular water remains in the cells and freezes, resulting in cell death. The rate of warming also affects the overall survival of the cells – rapid warming is preferred. Warming cells too slowly may allow recrystallization of ice within the cells. Rapid warming becomes especially important for recovery of cells which werefrozentooquickly(Bank,1973;Harris&Griffiths,1977;andMazur,1984). Careful control of both freezing and thawing is essential for successful cryopreservation.

This guide covers basic information on equipment, freeze media, and inventory systems, and also includes procedures for counting and freezing cells. The guidelines section provides information on how to customize the cryopreservation process for different cell types.

SAFety PRecAUtIOnSA face safety shield, lab coat, and low temperature protective gloves should be worn whenever handling vials or racks in a liquid nitrogen freezer. Improperly sealed vials can explode and produce projectiles of glass or plastic, vial contents, and liquid nitrogen. Replacement vials of precious cell cultures should be kept in another facility in case of a major disaster such as a fire or long term power outage.

The cryoprotectant dimethylsulfoxide (DMSO) is an irritant; and maybe harmful if inhaled, ingested, or absorbed through the skin. Avoiding anydirectcontactisthebestsafetyprecaution.DisposeoffreezemediumcontainingDMSOasdirectedbylocal,stateandfederallaws.

eQUIPMent AnD SUPPLIeS

A. FreezersLow temperatures of -150°C and below are required for successful long term storage. Cells can be stored at these temperatures for years if properly frozen. The viability of cells stored at temperatures above -130°C is significantly reduced,andisoftenonlyafewmonths(Mazur,1984;Shannon&Macy,1973). A security alarm for after-hours or weekends is useful for alerting key personnel in the event of temperature problems. Two types of freezers are primarily used for cryogenic storage. Mechanical freezers can maintain temperatures of -150°C, but require an electrical generator back-up in case of a power outage. Liquid nitrogen freezers range in temperature from 150°Catthetopofthefreezerto-196°Cintheliquidphaseatthebottomof the freezer. The liquid nitrogen levels should be monitored every few days to ensure these temperatures. Ice slush in the liquid nitrogen or ice on the lids and walls of either type of freezer can compromise the temperature. Minimizing the number of times freezers are opened, and periodic defrosting and cleaning are recommended for proper freezer operation.

B. VialsPlastic vials have gradually replaced glass ampules as the storage vessel ofchoice, the primary reason being safety. Hay has written a good protocol for those cell cultures which require the use of glass ampules (1978). However, glass ampules should not be used to freeze any biohazardous materials because of the danger of breakage and potential contamination of personnel, freezer, and other equipment (Schafer, et. al., 1976).Plastic vials come in a variety of sizes.The1 to2mLvials are ideal forstorage of 0.4 to 1.5 mL volumes of the cell suspension since they allow for uniform cooling and thawing vs. larger vials. The screw top vials are available with either external or internal screw threads (Figure 1).

Figure 1: Internally and Externally Threaded Vials

Internal Thread External Thread

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The external threads can help reduce contamination of the outside of the vial with the cell suspension, however, these vials usually cannot be stored in the liquid phase of the liquid nitrogen tank due to leakage around the cap. Internal threads allow the manufacturer to use O-ring gaskets for better sealing of the vials. However, any drops of liquid which lie on the internal threads may be pushed out of the vial when the cap is tightened, necessitating decontamination of the outside of the vial. Therefore, good aseptic technique and careful pipetting are important when transferring the cells to the vials (Figure 2).

Figure 2: Transferring Cells to Vials

The vials usually have an area for writing information about the contents withamarker.Foreasyretrievalandidentification,coloredcapsand/oranidentification number may be placed in the top of the vial (Figure 3).

Figure 3: Top View of Freezer Storage Box

Although the danger of explosion is reduced with plastic vials, liquid nitrogen escaping from around the caps can be hazardous, so laboratory personnel should wear protective face shields and low temperature protective gloves when handling vials in liquid nitrogen freezers.

cRyOPROtectAntS AnD FReeZe MeDIAThe cryoprotective agents DMSO and glycerol enhance the survival offrozen cells. This enhancement is achieved by decreasing the ice nucleation temperature (the temperature that ice forms). For instance, in mouse embryos, the ice nucleation temperature drops from -15°C in saline to-44°CineitherDMSOorglycerol(Rall,et.al.,1983).Thisreductionin nucleation temperature may take effect by: (1) maintaining the cell plasmamembrane’simpermeabilitytoextracellularice(Rule,et.al.,1980);(2) reducing or eliminating heterogeneous mechanisms of ice formation (Rasmussen&MacKenzie, 1972); or (3) keeping extracellular ice awayfrom the surface of the plasma membrane (Rall, et. al., 1983).

PRePARAtIOn OF ceLLSA. Trypan Blue Dye Exclusion Cell CountingCells with impermeable membranes (viable cells) will exclude dyes such as trypan blue, whereas non-viable cells will take-up the trypan blue and be visibly blue under the microscope.

Preparation of the 0.4% Trypan Blue Dye:1. Combine the following components in a bottle: • 0.4gTrypanbluedye • 0.85gNaCl,sodiumchloride • 100mLDeionizedordistilledwater2. Filter the mixture through Whatman no. 2 filter paper into a 100 mL

bottle.3. Label the bottle and store at room temperature.4.Thetrypanbluedyestainingsolutioncanbestoredforapproximately6

months.

PRePARAtIOn OF tHe ceLLS FOR cOUntIng1. When using a monolayer culture, prepare a single cell suspension in a salt

solution (ie. phosphate buffered saline or Hank’s Balanced Salt Solution). Suspension cultures can often be mixed with trypan blue dye d i r e c t l y , although growth medium salts and serum proteins stain blue and may give a high background. If this occurs, centrifuge the cells and resuspend them in a salt solution.

2.Mix0.5mLofthecellsuspensionand0.5mLof0.4%trypanbluedyein a small test tube.

3. Count the cells on the hemocytometer (see section B) within 15 minutes (viable cells may also take-up the dye after 15 minutes).

4. If the cells are not dispersed (ie. clumped), repeat the procedure after vigorous pipetting of the cell suspension. If the cell number is greater than 100 per square (1 mm2), then dilute the cell suspension appropriately.

Freeze Media

C

Freeze Media

C

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B. Counting Cells with a HemocytometerEach 1 mm x 1 mm square of a hemocytometer holds 0.0001 mL with the coverslip in place. With the coverslip lying on the chambers, use a pippette to transfer a small amount of the cell suspension to each of the two chambers.Allowthechamberstofillbycapillaryaction.Donotoverfillorunderfill the chambers or disturb the coverslip. A diagram of the grid pattern on the hemocytometer is shown in Figure 4.

Figure 4: Standard Hemocytometer Chamber

Viable Cell Counting:Todeterminethenumberofviablecells/mL,countthecellsfreeoftrypanblue dye in at least 4 squares for both chambers and divide by the total number of squares (1 mm2) counted. Count the cells on the outside edges of the square for only two of the four sides.

Viable Cells/mL = Average no. of cells/square x dilution factor x 104

Example: All of the fibroblast-like cells were removed from a culture flask with0.25%trypsinandresuspendedin10mLofbuffer.Asmall samplewas diluted 5-fold and then mixed 1:1 with trypan blue dye. The number of cells which excluded trypan blue dye in eight squares (1 mm2)was360.Therefore,thenumberofviablecells/mLequals4,500,000cells/mLandthetotal number of cells in the flask was 45,000,000.

Calculations: ViableCells/mL-360/8x5x2x104=4,500,000cells/mL

Percent Viability:By counting the number of viable cells (without trypan blue dye) and the dead cells (trypan blue dye inside the cells), the percent viability can be determined.

% Cell Viability = total viable cells (unstained ) x 200 total cells (stained and unstained)

gUIDeLIneS FOR FReeZIng AnD tHAWIng ceLLSThe following guidelines have been optimized for freezing murine hybridomas, human and murine myelomas, and fibroblast-like cells in ORIGEN®DMSO FreezeMedium. For optimal recovery,modificationson the concentrations of freeze medium components, equilibration time, cooling rate, and handling of the cells after thawing may be necessary.

A. Freezing Cells1.Protectivesafetyglassesandglovesarerecommendedforuseatalltimes.

When handling vials stored in liquid nitrogen, a full face shield and low temperature protective gloves should be worn (Hay, 1978).

2. Grow the cells until they are in log phase, approximately 5 x 105cells/mL for B-cell hybridomas. The cell viability, as assessed by trypan blue dye-exclusion(seeprevioussection),shouldbegreaterthan70%becauserecovery of viable cells after thawing is proportional to the pre-freeze cell viability (see Figure 5).

Figure 5: Recovery vs Initial Cell Viability

ORIGEN® is a registered trademark of IGEN, Inc.

1 mm

% Viable Pre-freeze Cells

SP2/0

% R

ecov

ery

of V

iabl

e C

ells

0 20 40 60 80 100

80

70

60

50

40

30

Several culturesofSP2/0myelomacellswithdifferentpercentagesofviable cells were frozen at 106 viable cells per vial. Each vial was later thawed and the percentage of viable cells recovered was determined.

PercentRecovery=no.ofviablecellsrecoveredafter thawing/106 x 100

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3. Calculate the volume of cells required to yield a density of 106 to 107cells/mL (for example, 10 mL of a culture at 5 x 105cells/mLisrequiredtoachieve 5 x 106 in 1 mL of freezing solution). Centrifuge the appropriate volume of cells to form a cell pellet (100 - 300 x g for 10–20 min). DiscardthesupernatantmediumandresuspendthecellswithORIGEN®DMSOFreezeMedium(thawedandatroomtemperature,17–25°C)orother suitable freeze solution.

4. Transfer the cell suspension to pre-labeled plastic cryovials (Simione, et. al., 1977). Before beginning the freezing process, the cells should equilibrate with the freeze medium for 5 minutes but no longer than 10 minutes, including the time it takes to resuspend and transfer the cells to the vials.

5. Freeze the cells at the appropriate cooling rate. Most cells can be cooled atarateof1°Cperminutesuntilat-30°C,andatafasterrate(5–20°C/min) to -100°C to -150°C with a programmable freezer and obtain good viabilityandrecovery(Farrant,et.al.,1977;Shannon&Macy,1973).Alternatively, the vials can be slowly frozen by placing the vials in a polystyrene or styrofoam box which is located at the bottom of a -55°C to-70°Ctemperaturefreezerfor4to24hours(Dougherty,1962).

B. Thawing Cells1. Before thawing the cells, prepare and warm the growth medium2.Pippettewarmedgrowthmedium(10xthecellsuspensionvolume)into

a sterile centrifuge tube. Repeat for the number of vials being thawed.3. Thaw the vial of cells quickly in a 37°C water bath. As soon as the ice has melted(about50–60seconds),removethevialfromthewaterbathanddecontaminatetheoutsideofthevialwith70%alcohol.

4. Aseptically transfer the vial contents to a sterile centrifuge tube containing growth medium (see step 2).

5. To remove the cryoprotectant, centrifuge the cells (100 x g for 10 min) and discard the supernatant liquid.

6.Resuspendthecellsinthegrowthmediumatapproximately1–2x105 viablecells/mLandtransferthecellsuspensiontoatissuecultureflask.

Alternate Methods:• Cellswhicharesusceptibletoosmoticshockmaybeslowlydiluted(over

2 – 5 minutes) with warm growth medium directly after thawing (step 3) (Strong,et.al.,1975;Strong,1976;Weiner,1976).

• Forcellsthatattachtothecultureflask,insteadofthecentrifugation(step5), transfer the diluted cells to a tissue culture flask and exchange the medium after 24 hours.

InVentORy SySteMSTo minimize the time spent locating vials in low temperature freezers, a good inventory system should be used. Computer spreadsheet software programs, such as Lotus 123, are useful for vial inventories, and can be invaluable for tracking vials of cells which are used over several years. Back-up disks with the inventory can easily be placed in fire-proof cabinets or boxes. A hard copy of the inventory is often easier to prepare but harder to store and back-up.

Since many people may want to access the freezer, a simple convention should be used to identify the vial contents and its location. A hard copy of the inventory should be updated as vials of cells are frozen or thawed. AsampleinventoryspreadsheetisshowninFigure6.

Figure 6: Sample Inventory Sheet

Another book containing information on the cells may also be useful for laboratories with substantial turnover (such as a university lab). The information can be noted on pre-printed worksheets, such as shown in Figure 7 and can be useful for tracking the viability of cell lines and the growth medium which was used before freezing. If the recovery of cells is decreasing with time, then another freeze lot of cells should be frozen for safe keeping.

SAMPLE

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ReFeRenceSBank, H. (1973). Cryobiology 10:157-170.

DiBenedetto,G.,J.Gill,M.T.Lopez-Vidriero,andS.W.Clarke(1989). Cryobiology26:328-332Dougherty,R.M.(1962).Nature198:550-552.

Farrant, J., C.A. Walter, H. Lee, and L.E. McGann (1977). Cryobiology 14:273-286.

Harris,L.W.andJ.B.Griffiths(1977).Cryobiology14:662-669.

Hay, R.J. (1978). Tissue Culture Manual 4:787-790.

Loretz,L.J.,A.P.Li,M.W.Flye,andA.G.E.Wilson(1989).Xenobiotica 19:489-498.

Mazur,P.(1984).Am.J.Physiol.247:125-142.

Rall,W.F.,P.Mazur,andJ.J.McGrath(1983).Biophys.J.41:1-12.

Rasmussen,D.H.,andA.P.MacKenzie(1972).WaterStructureatthe

Water-PolymerInterface(Jellinek,H.H.G.,Ed.)PlenumPublishing, NewYork,p.126-145.

Rule,G.S.,P.Law,J.Kruuv,andJ.R.Lepock(1980).J.Cell.Physiol. 103:407-416.

Schafer,T.W.,J.Everett,G.H.Silver,andP.E.Came(1976).Science 191:24-25.

Shannon, J.E., and M.L. Macy (1973). Tissue Culture Methods and

Applications(Kruse,P.F.,Jr.andM.K.Patterson,Jr.Eds.)AcademicPress, NewYork,p.712-718

Simione,F.P.Jr.,P.M.Daggett,M.S.McGrath,andM.T.Alexander(1977). Cryobiology 14:500-502

Strong,D.M.,J.N.Woody,M.A.Factor,A.Ahmed,andK.W.Sell (1975).Clin. Exp. Immunol. 21:442-455.

Strong,D.M.(1976).Trans.Proc.8:203-208

Weiner,R.S.(1976).J.Immunol.Met.10:49-60.

Figure 7: Preprinted Worksheet

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Cytogenetics and Diagnostic ProductsHeadline for TOCADAPtAtIOn OF ceLLS tO SeRUM-FRee cULtURe

Many cell lines grown in the presence of serum can be adapted to growth in serum-free medium using one of the following procedures.

A. Direct Adaptation MethodMany cell lines can be directly subcultured from medium containing serum to a serum-free medium. When the culture grown in the presence ofserumisinmid-logphasewithatleast90%viability,itcanbedilutedat a 1:2 or 1:3 ratio into fresh serum-free medium. This process should be repeated twice weekly until consistent growth is obtained. Initially cultures should be inoculated at a higher seeding density than what is normally used for subculturing due to significant loss of cells when directly seeded from serum-supplemented to serum-free medium. The cell growth rate is usually slower in serum-free medium for the first several passages before returning to the rates observed for cells in serum-supplemented medium. If this procedure is not successful, the sequential or weaning method should be used as described below.

B. Sequential Adaptation MethodCertain cell-types require a more gradual “weaning” process to achievegrowth under serum-free conditions. At each passage, the culture is diluted into a mixture of the serum-containing medium and the serum-free medium. Initially a 1:1 ratio of fresh serum-containing medium to serum-free medium can be used. With each subsequent passage, the relative amount of the serum-free medium is increased until complete independence of serum is achieved. At each passage the culture should be in mid-log growth and the dilution into fresh medium should be roughly at a 1:2 to 1:3 ratio. Cells should be subcultured twice per week. At each passage, a back-up flask should be seeded with a serum concentration known to be adequate to maintain cell viability inthe event that the new medium condition does not succeed.

For cell lines which are adherent in the presence of serum, adaptation to serum-free media will often result in the cultures becoming loosely adherent, possibly with clumping. The use of trypsin for passaging should be avoided as the level of serum in the fresh medium is reduced. For cells which are trypsinized, or where centrifugation and resuspension in fresh medium is usedforpassaging,theadditionofapproximately20%volumeofcell-freespent (conditioned) medium may aid in adaptation. If the cells do not thrive, or look unhealthy at any one particular stage of adaptation, maintain the cells for an additional passage in the previous stage medium ratio before subculturing into the next stage.

gUIDeLIneS FOR SeRUM-FRee cRyOPReSeRVAtIOn OF SeRUM-FRee ADAPteD ceLLS

A. Freezing Cells1.Preparesufficientcellsatlogphasegrowthtoyieldadensityof1-2E+07cells/mLoffreezingmediumforeachvialofcellsdesiredtobefrozen.

2.Determine the amount of freezing medium required and prepare bycombining cold fresh serum-freemediumof interestwith7%DMSOand sterile filter.

3. Collect and pellet the appropriate volume of cells by centrifugation at 300 x g for 3-5 minutes. Aspirate the supernatant medium and resuspend the cells in the appropriate volume of freezing medium.

4. Transfer 1mL of cell suspension into each sterile pre-labeled plastic cryovial, cap tightly and freeze accordingly.

5. Transfer frozen vials to liquid nitrogen storage.

B. Thawing CellsSame as techniques and alternative method previously described in our catalog.

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Irvine ScientificIndex

9144 ALPHAMEMEARLE’SSALTS 199142 ALPHAMEMEARLE’SSALTSWITHOUTNUCLEOSIDES 1991030-006 AMINOSTAT-FLM®-PG 1099473 CHANG AMNIO™ 1291031 CHANG MARROW™ 14C100 CHANGMEDIUM®BASAL 12C101 CHANGMEDIUM®BASAL 1291004 CHANGMEDIUM®BMC 14C106 CHANGMEDIUM®C,FROZENSUPPLEMENT 12C108 CHANGMEDIUM®C,FROZENSUPPLEMENT 1299419 CHANGMEDIUM®C,LYOPHILIZEDKIT 12T101-019 CHANGMEDIUM®C,LYOPHILIZEDKIT 12T101-059 CHANGMEDIUM®C,LYOPHILIZEDKIT 12T105 CHANGMEDIUM®D 1399404 CHANGMEDIUM®DW/GENTAMICIN 1391005 CHANGMEDIUM®MF 14T104 CHANGMEDIUM®,In Situ 139195 CLICK’SMEDIUM(EHAA) 179311 COLCEMID®SOLUTIONwithSEPTUMCAPS 219041 DEFICIENTDMEHIGHGLUCOSE 169031 DMEHIGHGLUCOSE 169024 DMEHIGHGLUCOSEwithoutL-GLUTAMINE 169314 EDTA(VERSENE)SOLUTION 223000A FETALBOVINESERUM 152014 FORMULAFEDBOVINECALFSERUM 159355 GENTAMICINSULFATESOLUTION 219056 HAM’SF-10 179058 HAM’S F-12 17

9052 HAM’SF-12/DMEHIGHGLUCOSE1:1MIXTURE 179077 HAM’SF-12K(KAIGHN’SNUTRIENTMIXTUREF-12) 179230 HBSS10X-HANKS’BALANCEDSALTSOLUTION 209220 HBSS1X-HANKS’BALANCEDSALTSOLUTION 209228 HBSS1X-HANKS’BALANCEDSALTSOLUTION 209319 HEPESBUFFERSOLUTION1M 2191006 ISGIANTCELLTUMOR-CONDITIONEDMEDIUM 219032 ISCOVE’SMODIFIEDDULBECCO’SMEDIUM 169317 L-GLUTAMINESOLUTION 219090 McCOY’SMEDIUM5A 179126 MEM1XEARLE’SSALTS 199130 MEM NEAA 1X EARLE’S SALTS 199154 MODIFIEDRPMI1640 189242 PBS10X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 209236 PBS1X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 209240 PBS1X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 2096691 PHYTOHEMAGGLUTININ-PHA(REAGENTGRADE) 219281 POTASSIUMCHLORIDESOLUTION0.075N 209160 RPMIMEDIUM1640 189159 RPMIMEDIUM16401XHEPES 189157 RPMIMEDIUM16401XHEPESW/L-GLUTAMINE 189161 RPMIMEDIUM1640W/L-GLUTAMINE 189342 TRYPSINEDTA10XSOLUTION 229340 TRYPSINEDTA1XSOLUTION 229341 TRYPSINEDTA1XSOLUTION 229336 TRYPSIN(1:250)12.5%10XSOLUTION 229309 WATERFORINJECTION(WFI)GRADEWATER* 21

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2014 FORMULAFEDBOVINECALFSERUM 159024 DMEHIGHGLUCOSEwithoutL-GLUTAMINE 169031 DMEHIGHGLUCOSE 169032 ISCOVE’SMODIFIEDDULBECCO’SMEDIUM 169041 DEFICIENTDMEHIGHGLUCOSE 169052 HAM’SF-12/DMEHIGHGLUCOSE1:1MIXTURE 179056 HAM’SF-10 179058 HAM’S F-12 179077 HAM’SF-12K(KAIGHN’SNUTRIENTMIXTUREF-12) 179090 McCOY’SMEDIUM5A 179126 MEM1XEARLE’SSALTS 199130 MEM NEAA 1X EARLE’S SALTS 199142 ALPHAMEMEARLE’SSALTSWITHOUTNUCLEOSIDES 199144 ALPHAMEMEARLE’SSALTS 199154 MODIFIEDRPMI1640 189157 RPMIMEDIUM16401XHEPESW/L-GLUTAMINE 189159 RPMIMEDIUM16401XHEPES 189160 RPMIMEDIUM1640 189161 RPMIMEDIUM1640W/L-GLUTAMINE 189195 CLICK’SMEDIUM(EHAA) 179220 HBSS1X-HANKS’BALANCEDSALTSOLUTION 209228 HBSS1X-HANKS’BALANCEDSALTSOLUTION 209230 HBSS10X-HANKS’BALANCEDSALTSOLUTION 209236 PBS1X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 209240 PBS1X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 209242 PBS10X-DULBECCO’SPHOSPHATEBUFFEREDSALINESOLUTION 209281 POTASSIUMCHLORIDESOLUTION0.075N 209309 WATERFORINJECTION(WFI)GRADEWATER* 21

9311 COLCEMID®SOLUTIONwithSEPTUMCAPS 219314 EDTA(VERSENE)SOLUTION 229317 L-GLUTAMINESOLUTION 219319 HEPESBUFFERSOLUTION1M 219336 TRYPSIN(1:250)12.5%10XSOLUTION 229340 TRYPSINEDTA1XSOLUTION 229341 TRYPSINEDTA1XSOLUTION 229342 TRYPSINEDTA10XSOLUTION 229355 GENTAMICINSULFATESOLUTION 2191004 CHANGMEDIUM®BMC 1491005 CHANGMEDIUM®MF 1491006 ISGIANTCELLTUMOR-CONDITIONEDMEDIUM 2191031 CHANG MARROW™ 1496691 PHYTOHEMAGGLUTININ-PHA(REAGENTGRADE) 2199404 CHANGMEDIUM®DW/GENTAMICIN 1399419 CHANGMEDIUM®C,LYOPHILIZEDKIT 1299473 CHANG AMNIO™ 123000A FETALBOVINESERUM 1591030-006 AMINOSTAT-FLM®-PG 10C100 CHANGMEDIUM®BASAL 12C101 CHANGMEDIUM®BASAL 12C106 CHANGMEDIUM®C,FROZENSUPPLEMENT 12C108 CHANGMEDIUM®C,FROZENSUPPLEMENT 12T101-019 CHANGMEDIUM®C,LYOPHILIZEDKIT 12T101-059 CHANGMEDIUM®C,LYOPHILIZEDKIT 12T104 CHANGMEDIUM®,In Situ 13T105 CHANGMEDIUM®D 13

nUMeRIcAL InDeXCatalog Number Description Page No.

Catalog Number Description Page No.

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