ATCC Primary Cell Culture Guide

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ATCC

PRIMARY CELL CULTURE GUIDE

tips and techniques for culturing primary cells

THE ESSENTIALS OF

LIFE SCIENCE RESEARCH

GLOBALLY DELIVERED


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Table of Contents

ATCC Primay Cell Solutions...........................................................................1

ATCC Primay Human Endothelial Cell Solutions.............................................5

ATCC Primay Human Smooth Muscle Cell Solutions.......................................9

ATCC Primay Human Epithelial Cell Solutions...............................................12

ATCC Primay Human Fibroblast Solutions.....................................................15

ATCC Primay Human Keatinocyte Solutions................................................19

ATCC Primay Human Melanocyte Solutions..................................................22

ATCC Human Mesenchymal Stem Cell and Differentiation Solutions..........25

References .......................................................................................................31


3/[email protected] Phone800.638.6597page 1

ATCCPRIMARY CELL SOLUTIONS

Guide to Culturing Human Primay Cels

Primary cells and cell types

Primay cell cultures more closely mimic the physiological state of cells in vivo and geneate morerelevant data representing living systems. Primay cultures consist of cells that have been freshlyderived from a living organism and are maintained for growth in vitro. Primay cells can be categorizedaccording to the genus from which they are isolated, as well as by species or tissue type. Eachmammalian tissue type is derived from the embyonic germ layer consisting of ectoderm, endodermand mesoderm, which differentiate into the many cell types that organize into tetiay structures suchas skin, muscle, internal organs, bone and catilage, the nevous system, blood and blood vessels1. Thecell types most frequently found in primay cell culture are epithelial cells, fibroblasts, keatinocytes,melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells.

Basic properties of primary cellsOnce adapted to in vitroculture conditions, primay cells undergo a limited, predetermined number ofcell divisions before entering senescence. The number of times a primay cell culture can be passagedis minimal due to the Haylick Limit, nutrient requirements and culture conditions, and the expetiseby which they are manipulated and subcultured. In contast, cell lines that have been immotalized byvial, hTERT or tumorigenic tansformation typically undergo unlimited cell division and have an infinitelifespan. And, unlike tumor cell lines cultured in medium containing 10% to 20% serum, primay cellcultures are fastidious, requiring optimized growth conditions, including the addition of tissue specificcytokines and growth factors for propagation in serum-free or low-serum growth media.

Benefits of primary cellsPrimay cell cultures are commonly used as in vitrotools for pre-clinical and investigative biologicalresearch, such as studies of inter- and intacellular communication, developmental biology, andelucidation of disease mechanisms, such as cancer, Parkinsons disease, and diabetes. Historically,investigators have employed immotalized cell lines in research related to tissue function; however, theuse of cell lines containing gross mutations and chromosomal abnormalities provides poor indicatorsof normal cell phenotype and progression of early-stage disease. The use of primay cells, maintainedfor only shot periods of time in vitro, now seves as the best representative of the main functionalcomponent of the tissue (in vivo) from which they are derived.

Isolation of primary cellsThe isolation and purification of peripheal blood cells can be easily achieved by differentialcentrifugation or by positive soting using magnetic beads. On the other hand, the isolation of a purepopulation of cells from primay tissue is often difficult to peform, and requires knowledge of howthe cell stata should be teased apat into a suspension containing only one predominant cell type.Diagam 1 is an illustation of some of the basic steps used to establish a primay cell culture14.

Primary cell culture

Growth requirements

Primay cells, except for those derived from peripheal blood, are anchoage-dependent, adherent

cells, meaning they require a suface in order to grow properly in vitro. In most cases, primay cells arecultured in a lat un-coated plastic vessel, but sometimes a microcarrier, which can greatly increase thesuface area, can be used. A complete cell culture media, composed of a basal medium supplemented


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with appropriate growth factors and cytokines, is required. During establishment of primay cultures, itmay be useful to include an antibiotic in the growth medium to inhibit contamination introduced fromthe host tissue. These may include a mixture of gentamicin, penicillin, streptomycin and amphotericinB. However, long-term use of antibiotics is not advised, since some reagents, such as amphotericin B,

may be toxic to cells over time.

Maintenance

The maintenance phase begins when cells have attached to the suface of the culture dish. Attachmentusually occurs about 24 hours after initiation of the culture. When initiating a culture of cyopresevedprimay cells, it is impotant to remove the spent media once the cells have attached because DMSOis harmful to primay cells and may cause a drop in post-thaw viability. When cells have reached adesired percent of cellular conluence and are actively prolifeating, it is time to subculture. It is bestto subculture primay cell cultures before reaching 100% conluence, since post-conluent cells mayundergo differentiation and exhibit slower prolifeation after passaging.

Cellular conluenceCellular conluence refers to the percentage of the culture vessel inhabited by attached cells. Forexample, 100% cellular conluence means the suface area is completely covered by cells, while 50%conluence means roughly half of the suface is covered. It is an impotant paameter to tack andassess in primay cell culture because different cell types require different conluence end points, atwhich point they need to be subcultured.

Levels of cellular conluence

Subculture

Anchoage-dependent cells grow in monolayers and need to be subcultured at regular intevals to

maintain exponential growth. Subculturing procedures, including recommended split-atios andmedium replenishment (feeding) schedules for each ATCC primay cell culture, are provided on the

Tissue Acquisition Dissection Disaggregation Incubation & GrowthSepaation &Purification

Process primaytissue, removing

fatty and necroticcells

Mechanicalor enzymatic

disaggregation. Enzymes used:

Typsin Collagenase II Elastase Hyaluronidase DNase

Incubate dispersedcells

Change medium 24hours after initiationto remove loosedebris & unattachedcells

Futher purificationof primay cells

achieved by: Selective media Remove cells at

different levels ofattachment

Immunomagneticbeads

Diagam 1. Basic steps used to isolate cells from primay tissue

Human smooth muscle cells between 20%and 30% conluence

Human melanocyte cells between 50%and 60% conluence

Human keatinocytes at nearly 100%conluence (note the formation ofvacuoles and differentiated cells)



product information sheet provided with each cell (available online at www.atcc.org). Subcultivation ofmonolayers involves the breakage of both inter- and intacellular cell-to-suface bonds. Most adherentprimay cells require the digestion of their protein attachment bonds with a low concentation of aproteolytic enzyme such as typsin/EDTA. After the cells have been dissociated and dispersed into asingle-cell suspension, they are counted and diluted to the appropriate concentation and tansferredto fresh culture vessels where they will reattach and divide.

Cell Counting

Hemocytometers are commonly used to estimate cell number and determine cell viability with the aidof an exclusion dye such as Typan Blue or Eythrosin B. A hemocytometer is a fairly thick glass slidewith two counting chambers, one on each side. Each counting chamber has a mirrored suface with a 3 3 mm grid consisting of 9 counting squares. The chambers have aised sides that will hold a coverslipexactly 0.1 mm above the chamber loor. Each of the 9 counting squares holds a volume of 0.0001 mL.Alternatively, counting cells can also be achieved by using an automated cell counter, such as Vi-CELL.

For a detailed description of cell counting using a hemocytometer, refer to the ATCC Animal Cell

Culture Guide: Tips and Techniques for Continuous Cell Lines available online at www.atcc.org.Cyopresevation and Recovey

Special attention is needed to cyopreseve and thaw primay cells in order to minimize cell damageand death during each process. Cyopresevation of human cells is best achieved with the use of acyoprotectant, such as DMSO or glycerol. Most primay cell cultures can be cyopreseved in a mixtureof 80% complete growth medium supplemented with 10% FBS and 10% DMSO. The freezing processshould be slow, at a ate of -1C per minute, to minimize the formation of ice cystals within the cells.Once frozen, cultures are stored in the vapor phase of liquid nitrogen, or below -130C. Additionalinformation about freezing cells can be found in ATCC Technical Bulletin No. 3: Cyogenic Presevationof Animal Cels, available online at www.atcc.org.

Thawing cyopreseved cells is a apid process and is accomplished by immersing frozen cells in a 37Cwater bath for about 1 to 2 minutes. Care should be taken not to centrifuge primay cells upon thaw,since they are extremely sensitive to damage during recovey from cyopresevation. It is best toplate cells directly upon thaw, and allow cultures to attach for the first 24 hours before changing themedium to remove residual DMSO.

Challenges of primay cell isolation and culture

There are seveal challenges associated with the use of primay cells. One of the greatest hurdlesprimay cell culturists face is limited cell accessibility due to issues with donor tissue supply, difficulty

with cell isolation/purification, quality assuance and consistency, and contamination risks. Datacompaability is also a serious issue with primay cell use, and arises out of variability among reagentsused and the procedures implemented by individual scientists and laboatories to isolate and cultureprimay cells. A comparison between primay cells and continuous cell lines is described in Table 1.



Table 1.Comparison between primay cells and continuous cell lines

Propeties Primay cells Continuous cell lines

Life span & cellprolifeation

Finite; limited to a small number of celldoublings

Infinite when handled properly

ConsistencyVariability exists between donors andprepaations Minimal variability

Genetic IntegrityRetains in vivotissue genetic makeupthrough cell doublings

Subject to genetic drift as cells divide

Biological relevanceMore closely mimics the physiology ofcells in vivo

Relevance can drift over time as cellsdivide

Ease of use (freeze-thaw& use)

Requires optimized culture conditionsand careful handling

Well established conditions and robustprotocols exist

Time & expense to use More time and less abundance of cells Less time and more abundance of cells

ATCC Primary Cell Solutions

ATCC has provided a solution to help investigators overcome the high cost and inconsistency foundin routine primay cell culture with the development of ATCCPrimay Cell Solutions, a standardizedcell culture system that includes high quality cells, media, supplements, reagents and protocols. ATCC

Primay Cell Solutions focuses on providing researchers with superior quality from a trusted source each lot of ATCC Primay Cell Solutions primay cells is:

Cyopreseved at early passage

ATCC Primay Cell Solutions primay cells are frozen at passages 1 through 3

Early passage material ensures higher viability and optimal plating efficiencyPeformance tested

ATCC Primay Cell Solutions cells, media, kit supplements, and reagents are tested for optimalreliability

Growth and morphology are assessed to ensure all components work synergistically

Quality controlled to ensure safety, purity, and functionality

Sterility testing - all cells are tested for bacteria, yeast, fungi, and Mycoplasma

Vial testing - HIV-1, HIV-2, HBV, and HCV tissue screening is peformed at isolation

Viability and Growth - viability and growth of each lot of cells is checked before freezing andafter-thawing

Staining - staining for cell-specific marker expression is determined and peformed for somecell types

ATCC Primay Cell Solutions basal media and cell-specific growth kits are designed to suppot recoveyand prolifeation of ATCC Primay Cell Solutions primay cells in vitro. Used as a system, ATCC PrimayCell Solutions primay cells, basal media and cell-specific growth kits provide all the componentsneeded to be successful in primay cell research. (Detailed formulations for each growth kit can befound at www.atcc.org.)



Endothelial Cells

[email protected] Phone800.638.6597page 5 [email protected] Phone800.638.6597

ATCC PRIMARYHUMANENDOTHELIALCELLSOLUTIONSIntroductionEndothelial cells form the endothelium - the thin layer of cells that linethe interior suface of blood vessels forming a smooth anticoagulant

suface that functions as a selective filter to regulate the passage ofgases, luid, immune cells and various molecules14. Human endothelialcells can be isolated from human umbilical vein, the aota, the pulmonayand coronay ateries, and the skin, and seve as useful tools in the studyof angiogenesis, cancer theapy, wound healing, burn theapy, high-throughput and high-content screening projects, cell signaling studies,gene expression profiling, toxicology screening, tissue engineering andregeneation.

ATCC Primay Human Endothelial Cells can be cultured in complete growth medium containing eitherbovine bain extact (BBE) or vascular endothelial growth factor (VEGF). Use of the Endothelial Cell

Growth Kit-VEGF (ATCC PCS-100-041) will suppot a faster ate of prolifeation, while the EndothelialCell Growth Kit-BBE (ATCC PCS-100-040) is recommended if a less defined cell culture medium isdesired.

Cell Culture ProtocolsMaterials Needed

Primay cells and complete growth medium

Endothelial Cells Growth Kit Options* Basal Medium

Umbilical Vein Endothelial Cells, Normal, Human(ATCC PCS-100-010)

Choose ONE:

Endothelial Cell Growth Kit-BBE (ATCC PCS-100-040)

Endothelial Cell Growth Kit-VEGF (ATCC PCS-100-041)

Vascular Cell Basal Medium(ATCC PCS-100-030)

Umbilical Vein Endothelial Cells, Normal,Human, Pooled (ATCC PCS-100-013)

Aotic Endothelial Cells; Normal, Human (ATCCPCS-100-011)

Coronay Atey Endothelial Cells, Normal,Human (ATCC PCS-100-020),

Pulmonay Atey Endothelial Cells, Normal,Human (ATCC PCS-100-022),

Dermal Microvascular Endothelial Cells, Normal,Human, Neonatal (ATCC PCS-110-010)

For Microvascalar Endothelial

Cells, Choose ONE:Microvascalar Endothelial CellGrowth Kit-BBE (ATCC PCS-110-040)

Microvascalar Endothelial CellGrowth Kit-VEGF (ATCC PCS-110-041)

Reagents for Subculture

D-PBS (ATCC 30-2200)

Typsin-EDTA for Primay Cells (ATCC PCS-999-003)

Typsin Neutalizing Solution (ATCC PCS-999-004)

*Phenol red and antibiotics may be added if desired, and are listed in the appendix.

Primay Dermal MicovascularEndothelial Cells, Normal, Human,Neonatal (ATCC PCS-110-010)



Endothelial Cells

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Prepaation of Complete Growth Media

1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.

2. Thaw the components of the growth kit just prior to adding them to the basal medium. It isnecessay to warm the L-glutamine component in a 37C water bath and shake to dissolve anyprecipitates prior to adding to the basal medium.

3. Obtain one bottle of Vascular Cell Basal Medium from cold stoage.

4. Decontaminate the external sufaces of all growth kit component vials and the basal mediumbottle by spaying them with 70% ethanol.

5. Using aseptic technique, and working in a laminar low hood or biosafety cabinet, tansfer thevolume of each growth kit component (volumes for each growth kit are provided on the productinformation sheet; the following table represents the Endothelial Cell Growth Kit-VEGF) to thebottle of basal medium using a sepaate sterile pipette for each tansfer.

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure ahomogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.

7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When storedunder these conditions, complete growth media is stable for 30 days.

Endothelial Cell Growth Kit-VEGF (ATCC PCS-100-041)

Component Volume Final Concentation

rh VEGF 0.5 mL 5 ng/mL

rh EGF 0.5 mL 5 ng/mL

rh FGF basic 0.5 mL 5 ng/mL

rh IGF-1 0.5 mL 15 ng/mL

L-glutamine 25.0 mL 10 mMHeparin sulfate 0.5 mL 0.75 Units/mL

Hydrocotisone hemisuccinate 0.5 mL 1 g/mL

Fetal Bovine Serum 10.0 mL 2%

Ascorbic acid 0.5 mL 50 g/mL

Handling Procedure for Frozen Cells and Initiation of Cultures

1. Refer to the batch specific information provided on the lastpage of the product information sheet for the total numberof viable cells recovered from each lot of ATCC Primay HumanEndothelial Cells.

2. Using the total number of viable cells, determine how muchsuface area can be inoculated to achieve an initial seedingdensity between 2,500 and 5,000 cells per cm2for most PrimayHuman Endothelial Cells offered by ATCC; the exception beingPrimay Dermal Microvascular Endothelial Cells, which shouldbe seeded at a density of 5,000 cells per cm2.

3. Prepare the desired combination of lasks. Add 5 mL of

complete growth media per 25 cm

2

of suface area. Placethe lasks in a 37C, 5% CO2, humidified incubator and allow the media to pre-equilibate to

tempeature and pH for 30 minutes prior to adding cells.

Primay Dermal Microvascular EndothelialCells dual stained for von Willebandfactor (green) as a marker for endothelialcells and smooth muscle -actin (red)



Endothelial Cells

[email protected] Phone800.638.6597page 7

4. While the culture lasks equilibate, remove one vial of ATCC Primay Human Endothelial Cells fromstoage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility ofcontamination, keep the O-ring and cap out of the water. Thawing should be apid (approximately1 to 2 minutes).

5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by

dipping in or spaying with 70% ethanol. All opeations from this point onward should be carriedout under strict aseptic conditions.

6. Add the appropriate volume of complete growth media [volume = (1 mL x number of lasks tobe seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, tansfer the cells fromthe cyovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do notcentrifuge.

7. Tansfer 1.0 mL of the cell suspension to each of the pre-equilibated culture lasks prepared insteps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Culture.Pipette seveal times,then cap and gently rock each lask to evenly distribute the cells.

8. Place the seeded culture lasks in the incubator at 37C with a 5% CO2atmosphere. Incubate for

at least 24 hours before processing the cells futher.

Maintenance

1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),warm only the volume needed in a sterile conical tube. Avoid warming complete growth mediamultiple times.

2. 24 hours after seeding, remove the cells from the incubator and view each lask under themicroscope to determine percent cellular conluence.

3. Carefully remove the spent media without disturbing the monolayer.

4. Add 5 mL of fresh, pre-warmed complete growth media per 25 cm2of suface area and return thelasks to the incubator.

5. After 24 to 48 hours, view each lask under the microscope to determine percent cellularconluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures havereached approximately 80% conluence, and are actively prolifeating (many mitotic figures arevisible), it is time to subculture. Subculture endothelial cells before reaching conluence; post-conluent primay endothelial cells may exhibit slower prolifeation after passaging.

Subculture

1. Passage Primay Human Endothelial Cells when cultures have reached approximately 80%conluence.

2. Warm both the Typsin-EDTA for Primay Cells (ATCC PCS-999-003) and the Typsin NeutalizingSolution (ATCC PCS-999-004) to room tempeature prior to dissociation. Warm complete growthmedium to 37C prior to use with the cells.

3. For each lask, carefully aspiate the spent media without disturbing the monolayer.

4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual taces ofserum.

5. Add pre-warmed typsin-EDTA solution (1 to 2 mL for evey 25 cm2) to each lask.

6. Gently rock each lask to ensure complete coveage of the typsin-EDTA solution over the cells,and then aspiate the excess luid off of the monolayer.

7. Obseve the cells under the microscope. When the cells pull away from each other and round up



Endothelial Cells

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(typically within 3 to 5 minutes), remove the lask from the microscope and gently tap it fromseveal sides to promote detachment of the cells from the lask suface.

8. When the majority of cells appear to have detached, quickly add an equal volume of TypsinNeutalizing Solution (ATCC PCS-999-004) to each lask. Gently pipette or swirl the culture toensure all of the typsin-EDTA solution has been neutalized.

9. Tansfer the dissociated cells to a sterile centrifuge tube and set aside while processing anyremaining cells in the lask.

10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the lask to collect any additional cells that might havebeen left behind.

11. Tansfer the cell/D-PBS suspension to the centrifuge tube containing the typsin-EDTA-dissociated cells.

12. Repeat steps 10 and 11 as needed until all cells have been collected from the lask.

13. Centrifuge the cells at 150 x g for 3 to 5 minutes.

14. Aspiate the neutalized dissociation solution from the cell pellet and resuspend the cells in 2 to

8 mL fresh, pre-warmed, complete growth medium.15. Count the cells and seed new lasks at a density of 2,500 to 5,000 cells per cm2; however, be sure

to seed Primay Dermal Microvascular Endothelial Cells at 5,000 cells per cm2.

16. Place newly seeded lasks in a 37C, 5% CO2, incubator for at least 24 to 48 hours before processing

the cells futher. Refer to Maintenancefor guidelines on feeding.



Smooth Muscle Cells


ATCC PRIMARYHUMANSMOOTHMUSCLECELLSOLUTIONS

IntroductionVascular tissue is made up of a diverse population of cell types,including endothelial cells, smooth muscle cells, pericytes,

fibroblasts, and other connective tissue cell types. Togetherthese cell types form tight junctions or connections, whichallow for permeability for both passive and active tanspotacross the vessel wall. Vascular smooth muscle cells make upthe smooth muscle layer of blood vessels and can be co-culturedwith vascular endothelium14. Smooth muscle cells isolated fromhuman ascending (thoacic) and descending (abdominal) aota,coronay atey, and pulmonay atey are useful for studyingvascular diseases such as thrombosis and atherosclerosis.

Cell Culture ProtocolsMaterials Needed


Smooth Muscle Cells Growth Kit Options* Basal Medium

Aotic Smooth Muscle Cells, Normal, Human(ATCC PCS-100-012)

Vascular Smooth Muscle CellGrowth Kit (ATCC PCS-100-042)

Vascular Cell Basal Medium(ATCC PCS-100-030)

Coronay Atey Smooth Muscle Cells, Normal,Human (ATCC PCS-100-021)

Pulmonay Atey Smooth Muscle Cells, Normal,

Human (ATCC PCS-100-023)Reagents for Subculture






1. Obtain one growth kit from the freezer; make sure that the

caps of all components are tight.2. Thaw the components of the growth kit just prior to adding

them to the basal medium. It is necessay to warm theL-glutamine component in a 37C water bath and shake todissolve any precipitates prior to adding to the basal medium.

3. Obtain one bottle of Vascular Cell Basal Medium from coldstoage.

4. Decontaminate the external sufaces of all growth kitcomponent vials and the basal medium bottle by spayingthem with 70% ethanol.

5. Using aseptic technique, and working in a laminar low hoodor biosafety cabinet, tansfer the volume of each growth kit

ATCC Primay Cell Solutions aoticsmooth muscle cells (ATCC PCS-100-012)dual stained for von Willeband factor(green) as a marker for endothelial cells

and smooth muscle -actin (red) afterdifferentiation.

Primay Aotic Smooth Muscle Cells,Normal, Human (ATCC PCS-100-012)


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Smooth Muscle Cells


multiple times.

2. 24 to 36 hours after seeding, remove the cells from the incubator and view each lask under themicroscope to determine percent cellular conluence.



5. After 24 to 48 hours, view each lask under the microscope todetermine percent cellular conluence. If not ready to passage,repeat steps 3 and 4 as described above. When cultures have reachedapproximately 80% to 90% conluence, and are actively prolifeating(many mitotic figures are visible), it is time to subculture. Vascularsmooth muscle cells are contact inhibited; post-conluent vascularsmooth muscle cells may not prolifeate after passaging.

Subculture

1. Passage normal vascular smooth muscle cells when the culture has reached approximately 80%conluence.

2. Warm both the Typsin-EDTA for Primay Cells (ATCC PCS-999-003) and the Typsin NeutalizingSolution (ATCC PCS-999-004) to room tempeature prior to dissociation. Warm complete growthmedium to 37C prior to use with the cells.


4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual taces ofserum.



7. Obseve the cells under the microscope. When the cells pull away from each other and round up(typically within 1 to 3 minutes), remove the lask from the microscope and gently tap it fromseveal sides to promote detachment of the cells from the lask suface.

8. When the majority of cells appear to have detached, quickly add an equal volume of TypsinNeutalizing Solution (ATCC PCS-999-004) to each lask. Gently pipette or swirl the culture toensure all of the typsin-EDTA solution has been neutalized.

9. Tansfer the dissociated cells to a sterile centrifuge tube and set aside while processing anyremaining cells in the lask.

10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the lask to collect any additional cells that might havebeen left behind.




14. Aspiate the neutalized dissociation solution from the cell pellet and resuspend the cells in 2 to8 mL fresh, pre-warmed, complete growth medium.

15. Count the cells and seed new lasks at a density of 2,500 to 5,000 cells per cm

2

.16. Place newly seeded lasks in a 37C, 5% CO2, incubator for at least 24 to 48 hours before processing


Note:Cells are typically ready topassage after 7 to 9 days inculture when inoculated with2,500 cells/cm2.



Epithelial Cells

ATCC PRIMARYHUMANEPITHELIALCELLSOLUTIONS

IntroductionEpithelia are tissues found throughout the body. They line thecavities of glands and organs in the body and also cover lat sufaces,

such as skin. Epithelial cells are polarized cells that discriminatebetween an apical and basolateal compatment. They peforma variety of functions depending on their location, includingbounday and protection, sensoy, secretion, tanspotation,absorption and diffusion. One challenging aspect of culturingprimay epithelial cells is overgrowth of stromal cells. Stromalfibroblasts can be inhibited by culturing explants in low serum or serum-free culture media, limitingthe calcium concentation in the growth medium, selectively inhibiting fibroblasts with agents, orby targeting with antimesodermal antibodies14. ATCC Primay Cell Solutions offers epithelial cellsisolated from human bronchi, tachea and small aiways, as well as the cornea, prostate and kidneys.

The usefulness of such cultures has been found in studies related to inlammation, microbial infectionand pathogenesis including inluenza, cancer, toxicology, gene regulation and tissue development,cell-matrix inteactions, as well as application in toxicology testing and drug screening/development.

Cell Culture ProtocolsMaterials NeededPrimay cells and complete growth medium

Epithelial Cells Growth Kit Options* Basal Medium

Small Aiway Epithelial Cells; Normal, Human(ATCC PCS-301-010)

Small Aiway Epithelial CellGrowth Kit (ATCC PCS-301-

040) Aiway Epithelial Cell BasalMedium (ATCC PCS-300-030)Bronchial/Tacheal Epithelial Cells; Normal,

Human (ATCC PCS-300-010)

Bronchial Epithelial CellGrowth Kit (ATCC PCS-300-040)

Renal Proximal Tubule Epithelial Cells; Normal,Human (ATCC PCS-400-010)

Renal Epithelial Cell Growth Kit(ATCC PCS-400-040)

Renal Epithelial Cell BasalMedium (ATCC PCS-400-030)

Renal Cotical Epithelial Cells; Normal, Human(ATCC PCS-400-011)

Renal Mixed Epithelial Cells; Normal, Human(ATCC PCS-400-012)

Prostate Epithelial Cells; Normal, Human (ATCCPCS-440-010)

Prostate Epithelial Cell GrowthKit (ATCC PCS-440-040)

Prostate Epithelial CellBasal Medium (ATCC PCS-440-030)

Corneal Epithelial Cells; Normal, Human (ATCCPCS-700-010)

Corneal Epithelial Cell GrowthKit (ATCC PCS-700-040)

Corneal Epithelial Cell BasalMedium (ATCC PCS-700-030)






Primay Prostate Epithelial Cells; Normal,Human (ATCC PCS-440-010)



Epithelial Cells


1. Obtain one Epithelial Cell Growth Kit (corresponding to the epithelial cell type being used) fromthe freezer; make sure that the caps of all components are tight.

2. Thaw the components of the growth kit just prior to adding them to the basal medium.

3. Obtain one bottle of Epithelial Cell Basal Medium (corresponding to the epithelial cell being used)from cold stoage.


5. Using aseptic technique and working in a laminar low hood or biosafety cabinet, tansfer theindicated volume of each growth kit component as indicated in the cultures correspondingproduct information sheet, to the bottle of basal medium using a sepaate sterile pipette foreach tansfer.




1. Refer to the batch specific information provided on the last page of the product informationsheet for the total number of viable cells recovered from each lot of ATCC Primay HumanEpithelial Cells.

2. Using the total number of viable cells, determine how much suface area can be inoculated toachieve an initial seeding density of 5,000 cells per cm2.

3. Prepare the desired combination of lasks. Add 5 mL of complete growth medium per 25 cm2of

suface area. Place the lasks in a 37C, 5% CO2, humidified incubator and allow the media to pre-equilibate to tempeature and pH for 30 minutes prior to adding cells.

4. While the culture lasks equilibate, remove one vial of ATCC Primay Human Epithelial Cells fromstoage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility ofcontamination, keep the O-ring and cap out of the water. Thawing should be apid (approximately1 to 2 minutes).

5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate bydipping in or spaying with 70% ethanol. All opeations from this point onward should be carriedout under strict aseptic conditions.

6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of lasksto be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, tansfer the cells fromthe cyovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do notcentrifuge.

7. Tansfer 1.0 mL of the cell suspension to each of the pre-equilibated culture lasks prepared insteps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Culture.Pipette seveal times,then cap and gently rock each lask to evenly distribute the cells.

8. Place the seeded culture lasks in the incubator at 37C, 5% CO2atmosphere. Incubate for at least

24 hours before processing the cells futher.

Maintenance

1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),



Epithelial Cells

warm only the volume needed in a sterile conical tube. Avoid warming complete growth mediamultiple times.



4. Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm2of suface area and returnthe lasks to the incubator.

5. After 24 to 48 hours, view each lask under the microscope to determine percent cellularconluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures havereached about 80% conluence, and are actively prolifeating (many mitotic figures are visible), itis time to subculture.

Subculture

1. Passage normal human epithelial cells when the culture has reached about 80% conluence.

2. Warm both the Typsin-EDTA for Primay Cells (ATCC PCS-999-003) and the Typsin NeutalizingSolution (ATCC PCS-999-004) to room tempeature prior to dissociation. Warm the completegrowth medium to 37C prior to use with the cells.


4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.




8. When the majority of cells appear to have detached, quickly add an equal volume of the TypsinNeutalizing Solution (ATCC PCS-999-004) to each lask. Gently pipette or swirl the culture toensure all of the typsin-EDTA solution has been neutalized.

9. Tansfer the dissociated cells to a sterile centrifuge tube and set aside while processing anyremaining cells in the culture lask.

10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture lask to collect any additional cells thatmight have been left behind.

11. Tansfer the cell/D-PBS suspension to the centrifuge tube containing the typsin-EDTA-

dissociated cells.12. Repeat steps 10 and 11 as needed until all cells have been collected from the lask.


14. Aspiate neutalized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mLfresh, pre-warmed, complete growth medium.

15. Count the cells and seed new culture lasks at a density of 5,000 viable cells per cm2.

16. Place newly seeded lasks in a 37C, 5% CO2incubator for at least 24 to 48 hours before processing




Fibroblasts


ATCC PRIMARYHUMANFIBROBLASTSOLUTIONS

IntroductionFibroblasts play an impotant role in maintaining the structualintegrity of connective tissue, and synthesize extacellular

matrix proteins such as collagens, glycosaminoglycans, andglycoproteins15,16. Fibroblasts are morphologically heterogeneous,and their appeaance is dependent on in vivolocation and activity.Injuy of tissue displays a prolifeative stimulus for fibroblasts andinduces them to produce wound healing proteins. Fibroblasts usedin primay cell culture are commonly isolated from the dermislayer of human neonatal foreskin or adult skin. They are frequentlyused in studies related to wound healing, tissue engineering andregeneation applications, and the induction of pluripotent stem(iPS) cells. Fibroblasts, treated with mitomycin C to inhibit prolifeation, have been extensively used as

feeder layers to enhance the cultivation of human stem cells and keatinocytes in vitro.

ATCC Primay Human Fibroblasts can be cultured in complete growth medium with or without serum.The use of Fibroblast Growth KitSerum-Free creates a completely defined medium for the serum-freeculture of human fibroblasts. The ate of prolifeation is equal to or greater than media supplementedusing FBS (at concentations anging from 2% to 10%) through 10 population doublings.

Cell Culture Protocols

Materials NeededPrimay cells and complete growth medium

Fibroblasts Growth Kit Options* Basal MediumDermal Fiboblasts; Normal, Human Neonatal(ATCC PCS-201-010)

Choose ONE:Fibroblast Growth Kit SerumFree (ATCC PCS-201-040)

Fibroblast Growth Kit LowSerum (ATCC PCS-201-041)

Fibroblast Basal Medium(ATCC PCS-201-030)

Dermal Fiboblasts; Normal, Human, Adult(ATCC PCS-201-012)

Dermal Fiboblasts; Normal, Human Neonatal,Mitomycin C Treated (ATCC PCS-201-011)**



Typsin-EDTA for Primay Cells (ATCC PCS-999-003)Typsin Neutalizing Solution (ATCC PCS-999-004)

*Phenol red and antibiotics may be added if desired, and are listed in the appendix.**0.1% Gelatin Solution (ATCC PCS-999-027) is also needed for culturing Mitomycin C Treated Dermal Fibroblasts, Normal,Human Neonatal (ATCC PCS-201-011).


1. Obtain one growth kit from the freezer; make sure that the caps of all containers are tight.

2. Thaw the components of the growth kit just prior to adding them to the basal medium. It isnecessay to warm the L-glutamine component in a 37C water bath, and shake to dissolve anyprecipitates prior to adding to the basal medium.

3. Obtain one bottle of Fibroblast Basal Medium from cold stoage.

Primay adult human dermal fibroblasts(ATCC PCS-201-012) cultured in serum-free medium.



Fibroblasts

www.atcc.org page 16


5. Using aseptic technique and working in a laminar low hood or biosafety cabinet, tansfer thevolume of each growth kit component, as indicated in the following tables, to the bottle of basalmedium using a sepaate sterile pipette for each tansfer.

Fibroblast Growth KitSerum-Free (ATCC PCS-201-040)Component Volume Final Concentation

L-glutamine 18.75 mL 7.5 mM

Hydrocotisone Hemisuccinate 0.5 mL 1 g/mL

HLL Supplement 1.25 mLHSA 500 g/mLLinoleic Acid 0.6 MLecithin 0.6 g/mL

rh FGF 0.5 mL 5 ng/mL

rh EGF / TGF -1 Supplement 0.5 mL5 ng/mL30 pg/mL

rh Insulin 0.5 mL 5 g/mL


Fibroblast Growth KitLow Serum (ATCC PCS-201-041)


rh FGF 0.5 mL 5 ng/mL

L-glutamine 18.75 mL 7.5 mM


Hydrocotisone Hemisuccinate 0.5 mL 1 g/mL

rh Insulin 0.5 mL 5 g/mL

Fetal Bovine Serum 10.0 mL 2%

6. Tightly cap the bottle of complete growthmedium and swirl the contents gently to assure ahomogeneous solution. Do not shake forcefully toavoid foaming. Label and date the bottle.

7. Complete growth media should be stored in the dark

at 2C to 8C (do not freeze). When stored underthese conditions, complete growth media is stablefor 30 days.


1. Refer to the batch specific information provided on the last page of the product informationsheet for the total number of viable cells recovered from each lot of ATCC Primay HumanFibroblasts.

2. Using the total number of viable cells repoted, determine how much suface area can beinoculated to achieve an initial seeding density of 2,500 to 5,000 cells per cm2 for untreated

fibroblasts. Note: Mitomycin C treated fibroblasts should be seeded at a density of 20,000 to40,000 cells per cm2for use with stem cells.

Note:Gelatin-Coated Tissue Culture Flasks are neededto culture Mitomycin C Treated Dermal Fibroblasts(ATCC PCS-201-011) in order to achieve optimalcell attachment. Culture lasks should be preparedbefore thawing cells. Non-coated tissue culture

lasks can also be used, if needed.



Fibroblasts


3. Prepare the desired combination of lasks. Add 5mL of complete growth medium per 25 cm2ofsuface area. Place the lasks in a 37C, 5% CO

2, humidified incubator and allow the media to pre-

equilibate to tempeature and pH for 30 minutes prior to adding cells.

4. While the culture lasks equilibate, remove one vial of corresponding ATCC Primay HumanFibroblasts from stoage and thaw the cells by gentle agitation in a 37C water bath. To reduce

the possibility of contamination, keep the O-ring and cap out of the water. Thawing should beapid (approximately 1 to 2 minutes).


6. Add the appropriate volume of complete growth media [volume = (1 mL x number of lasks tobe seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, tansfer the cells fromthe cyovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do notcentrifuge.

7. Tansfer 1 mL of the cell suspension to each of the pre-equilibated culture lasks prepared in

steps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Cultures.Pipette seveal times,then cap and gently rock each lask to evenly distribute the cells.

8. Place the seeded culture lasks in the incubator at 37C, 5% CO2atmosphere. Incubate at least 24

hours before processing the cells futher.

Maintenance





5. After 24 to 48 hours, view each lask under the microscope to determine percent cellularconluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures havereached 80% to 100% conluence, and are actively prolifeating (many mitotic figures are visible),it is time to subculture. Fibroblasts are not a contact inhibited cell type.

Subculture1. Passage normal human fibroblasts when the cells have reached

approximately 80% to 100% conluence and are activelyprolifeating.

2. Warm both the Typsin-EDTA for Primay Cells (ATCC PCS-999-003) and the Typsin Neutalizing Solution (ATCC PCS-999-004)to room tempeature prior to dissociation. Warm the complete growth medium to 37C prior touse with the cells.


4. Rinse the cell layer two times with 3 to 5 mL of D-PBS per 25 cm2of suface area (ATCC 30-2200)to remove any residual taces of serum. Rinse the cell layer one time with 3 to 5 mL of D-PBS if

Note:Mitomycin C treated fibroblastsare mitotically arrested andcannot be subcultured.



Fibroblasts


serum-free culture conditions are used.



7. Obseve the cells under the microscope. When the cells pull away from each other and round up(typically within about 3 to 5 minutes), remove the lask from the microscope and gently tap itfrom seveal sides to promote detachment of the cells from the lask suface.

8. When the majority of cells appear to have detached, quickly add to each lask, a volume of theTypsin Neutalizing Solution (ATCC PCS-999-004) equal to the volume of typsin-EDTA solutionused previously. Gently pipette or swirl the culture to ensure all of the typsin-EDTA solution hasbeen neutalized.


10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture lask to collect any additional cells that

might have been left behind.11. Tansfer the cell/D-PBS suspension to the centrifuge tube containing the typsin-EDTA-

dissociated cells.



14. Aspiate the neutalized dissociation solution from the cell pellet and resuspend the cells in 2 to8 mL fresh, pre-warmed, complete growth medium.

15. Count the cells and seed new culture lasks at a density of 2,500 to 5,000 cells per cm2.



Inoculation of Mitomycin-C-treated Fibroblast Feeder Layers with Co-culture Cells of Interest

1. Aspiate the complete fibroblast growth medium from the feeder cell layer.

2. Add pre-warmed medium appropriate for the cell type of interest (e.g., embyonic stem cell).

3. Return the culture lask to the incubator at 37C, 5% CO2. Allow to equilibate for at least 30

minutes before seeding the cells of interest.

For more information regarding the use of feeder cells with stem cell lines, refer toATCCHuman ES/iPS Cell Culture Guide: Protocos for Feeder-free and Feeder-dependent Systemsavailable

online at www.atcc.org.



Keatinocytes


ATCC PRIMARYHUMANKERATINOCYTESOLUTIONS

IntroductionKeatinocytes are the most common type of skin cells making upthe majority of the epidermis. Dividing keatinocytes produce

keatin (a protein that provides strength to skin, hair and nails) andmigate to the suface of the skin, the statum corneum, where theyseve as the most abundant cells in the skins outermost layer17.Once present in the statum corneum, keatinocytes differentiate,stop dividing as cornified cells18and eventually undergo apoptosis.Keatinocytes can be isolated from different locations of the body.However, the most utilized keatinocytes in primay cell culturehave been isolated from the epidermis of human juvenile foreskinor human adult skin, the latter of which is significant in the study of adult diseases such as psoriasisand skin cancer14. Keatinocytes cultured in serum-free, low calcium growth medium will maintain the

cells in an un-differentiated, prolifeative state while inhibiting fibroblast outgrowth. Keatinocytesare useful for a number of scientific applications including the study of growth factor behavior, woundhealing, toxicity/irritancy studies, and use as target cells for derivation of induced pluripotent stemcells.


Materials NeededPrimay cells and complete growth medium

Keatinocytes Growth Kit Options* Basal Medium

Epidermal Keatinocyte; Normal, Human,Neonatal Foreskin (ATCC PCS-200-010) Keatinocyte Growth Kit

(ATCC PCS-200-040)Dermal Cell Basal Medium(ATCC PCS-200-030)Epidermal Keatinocyte; Normal, Human, Adult

(ATCC PCS-200-011)






Prepaation of Complete Growth Media1. Obtain one Keatinocyte Growth Kit from the freezer; make sure that the caps of all components

are tight.


3. Obtain one bottle of Dermal Cell Basal Medium from cold stoage.


5. Using aseptic technique and working in a laminar low hood or biosafety cabinet, tansfer theindicated volume of each growth kit component, as indicated in the following table, to the bottleof basal medium using a sepaate sterile pipette for each tansfer.

Primay Epidermal Keatinocytes; Normal,Human, Adult (ATCC PCS-200-011)



Keatinocytes


Keatinocyte Growth Kit (ATCC PCS-200-040)


Bovine Pituitay Extact (BPE) 2.0 mL 0.4%

rh TGF- 0.5 mL 0.5 ng/mL

L-Glutamine 15.0 mL 6 mMHydrocotisone Hemisuccinate 0.5 mL 100 ng/mL

rh Insulin 0.5 mL 5 g/ml

Epinephrine 0.5 mL 1.0 M

Apo-Tansferrin 0.5 mL 5 g/ml


7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When stored

under these conditions, complete growth media is stable for 30 days.


1. Refer to the batch specific information provided on the last page of the product informationsheet for the total number of viable cells recovered from each lot of ATCC Primay HumanKeatinocytes.

2. Using the total number of viable cells, determine how much suface area can be inoculated toachieve an initial seeding density between 2,500 and 5,000 cells per cm2.

3. Prepare the desired combination of lasks. Add 5 mL of complete growth medium per 25 cm2ofsuface area. Place the lasks in a 37C, 5% CO


equilibate to tempeature and pH for 30 minutes prior to adding cells.4. While the culture lasks equilibate, remove one vial of ATCC Primay Human Keatinocytes from

stoage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility ofcontamination, keep the O-ring and cap out of the water. Thawing should be apid (approximately1 to 2 minutes).



7. Tansfer 1.0 ml of the cell suspension to each of the pre-equilibated culture lasks prepared insteps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Culture.Pipette seveal times,then cap and gently rock each lask to evenly distribute the cells.



Maintenance

1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),warm only the volume needed in a sterile conical tube. Avoid warming complete growth media



Keatinocytes


multiple times.




5. After 24 to 48 hours, view each lask under the microscope to determine percent cellularconluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures havereached approximately 80% conluence, and are actively prolifeating (many mitotic figures arevisible), it is time to subculture. Keatinocytes will begin to terminally differentiate once they become100% conluent.

Subculture

1. Passage normal keatinocytes when the culture has reached approximately 70% to 80%conluence.






7. Obseve the cells under the microscope. When the cells pull away

from each other and round up (typically within 3 to 6 minutes),remove the lask from the microscope and gently tap it fromseveal sides to promote detachment of the cells from the lasksuface.









16. Place newly seeded lasks in a 37C, 5% CO2incubator for at least 24 to 48 hours before processingthe cells futher. Refer to Maintenancefor guidelines on feeding.

Note:If cells are difficult to detach,incubate each lask containingcells and the typsin-EDTAsolution at 37C to facilitatedispersal.



Melanocytes


ATCC PRIMARYHUMANMELANOCYTESOLUTIONS

IntroductionMelanocytes are found mainly in the epidermis, but may occurelsewhere in the body such as in the matrix of the hair. They are

specialized skin cells that produce the pigment melanin, whichgives skin its color and protects it from the hazardous effects ofUV adiation. The most useful melanocytes for research are isolatedfrom the epidermis of human juvenile foreskin or human adult skin.Special care must be taken to prevent contamination of melanocytecultures with keatinocytes or fibroblasts found in the epidermis.Lowering the concentation of calcium in the medium to 60 M andselecting for adherent melanocytes in hormone-supplementedmedium aids in the isolation of melanocytes from epidermal tissue14. Melanocytes are frequently usedin the in vitrostudy of wound healing, and as testing models for toxicity/irritancy studies, melanoma,

dermal response to UV adiation, psoriasis and other skin diseases, and cosmetic research (e.g., skinlightening compounds, skin protecting compounds).


Materials Needed


Melanocytes Growth Kit Options Basal Medium

Epidermal Melanocytes; Normal, Human,Neonatal (ATCC PCS-200-012)

Melanocyte Growth Kit (ATCCPCS-200-041) Dermal Cell Basal Medium

(ATCC PCS-200-030)Epidermal Melanocytes; Normal, Human, Adult(ATCC PCS-200-013) Adult Melanocyte Growth Kit(ATCC PCS-200-042)







1. Obtain one Melanocyte Growth Kit (corresponding to the melanocyte being used) from thefreezer; make sure that the caps of all components are tight.


3. Obtain one bottle of Dermal Cell Basal Medium from cold stoage.


5. Using aseptic technique and working in a laminar low hood or biosafety cabinet, tansfer theindicated volume of each growth kit component, as indicated in the following table, to the bottle

of basal medium using a sepaate sterile pipette for each tansfer.

Adult human melanocytes (ATCC PCS-200-013).



Melanocytes


Melanocyte Growth Kit (ATCC PCS-200-041)


rh Insulin 0.5 mL 5 g/ml

Ascorbic Acid 0.5 mL 50 g/ml

L-Glutamine 15.0 mL 6 mMEpinephrine 0.5 mL 1.0 M

Calcium Chloride 80 l 0.2 mM

M8 Supplement 5 ml Proprietay formulation



Handling Procedure for Frozen Cells and Initiation of Cultures1. Refer to the batch specific information provided on the last page of the product information

sheet for the total number of viable cells recovered from each lot of ATCC Primay HumanMelanocytes.


3. Prepare the desired combination of lasks. Add 5 mL of complete growth medium per 25 cm2ofsuface area. Place the lasks in a 37C, 5% CO



4. While the culture lasks equilibate, remove one vial of ATCC Primay Human Melanocytes fromstoage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility ofcontamination, keep the O-ring and cap out of the water. Thawing should be apid (approximately1 to 2 minutes).


6. Add the appropriate volume of complete growth medium [volume = (1 mL x number of lasksto be seeded) 1 mL] into a sterile conical tube. Using a sterile pipette, tansfer the cells fromthe cyovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not

centrifuge.7. Tansfer 1.0 ml of the cell suspension to each of the pre-equilibated culture lasks prepared insteps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Culture.Pipette seveal times,then cap and gently rock each lask to evenly distribute the cells.



Maintenance

1. Before beginning, pre-warm complete growth media in a 37C water bath. This will take between10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less),

warm only the volume needed in a sterile conical tube. Avoid warming complete growth mediamultiple times.



Melanocytes


2. 24 to 36 hours after seeding, remove the cells from the incubator and view each lask under themicroscope to determine percent cellular conluence.



5. After 24 to 48 hours, view each lask under the microscope to determine percent cellularconluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures havereached 80% to 90% conluence, and are actively prolifeating (many mitotic figures are visible),it is time to subculture. Melanocytes are not contact inhibited; however, they will prolifeate bestif the cells are passaged prior to 100% conluence. Melanocytes from lightly pigmented tissueshould reach 80% conluence in 7 to 9 days.

Subculture

1. Passage normal melanocytes when the culture has reached approximately 80% to 90% conluence.

2. Warm both the Typsin-EDTA for Primay Cells (ATCC PCS-999-003) and the Typsin Neutalizing

Solution (ATCC PCS-999-004) to room tempeature prior to dissociation. Warm the completegrowth medium to 37C prior to use with the cells.


4. Rinse the cell layer two times with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.



7. Obseve the cells under the microscope. When the cells pull awayfrom each other and round up (typically within 1 to 3 minutes),

remove the lask from the microscope and gently tap it fromseveal sides to promote detachment of the cells from the lasksuface.



10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture lask to collect any additional cells that

might have been left behind.11. Tansfer the cell/D-PBS suspension to the centrifuge tube containing the typsin-EDTA-dissociated cells.







Note:

Melanocytes are sensitive to over-typsinization.



Human Mesenchymal Stem Cells

page 25 [email protected] [email protected] Phone800.638.6597page 25

ATCC HUMANMESENCHYMALSTEMCELLAND

DIFFERENTIATIONSOLUTIONS

Introduction

ATCC Human Mesenchymal Stem Cells (MSCs) are self-renewingmultipotent adult stem cells. MSCs are taditionally found in thebone marrow, but can also be isolated from other tissues includingadipose, human umbilical cord or cord blood, and periphealblood. Adipose-derived mesenchymal stem cells are isolated fromhuman adipose (fat) tissues by lipoaspiation or biopsy. Umbilicalcord derived mesenchymal stem cells are isolated from WhatonsJelly found in human umbilical cord. Although MSCs from primayadipose tissue and Whatons Jelly are considered to be relativelyeasy to obtain, isolation of consistent stem cell populations from

such material is costly and time-consuming. Lipoaspiates and umbilical cord tissue represent aheterogeneous mixture of cell types, including adipocytes, endothelial cells, smooth muscle, pericytesand progenitor cells2. The inherent heterogeneity of this source material, then, yields a high potentialfor cellular contamination to predominate cultures.

To ensure the purity of ATCC Primay Cell Solutions Normal Human Mesenchymal Stem Cells, eachbatch is isolated from single-donor tissue, cyopreseved in the second passage, and tested for:

1. Authentication of growth and morphology, including adherence to plastic when cultured inoptimized growth medium consisting of Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030) supplemented with Mesenchymal Stem Cell Growth Kit-Low Serum (ATCC PCS-500-040)

2. Verification of suface antigen expression13, including a total of 10 markers: Positive ( 95%) forCD29, CD44, CD73, CD90, CD105, and CD166; and, negative (2%) for CD14, CD31, CD34, andCD45

3. Confirmation of multi-lineage differentiation into osteoblasts, adipocytes, and chondrocytesusing optimized differentiation kits and protocols

MSCs are useful tools for non-controversial stem cell differentiation research and for the creation ofinduced pluripotent stem (iPS) cell lines8. MSCs have also found applications in tissue engineering,3,14,5,11,cell theapy12, and regeneative medicine6,7,9,10.

Adipose-Derived Mesenchymal StemCells, Normal, Human (ATCC PCS-500-011).

0%

20%

40%

60%

80%

100%

120%

CD29 CD44 CD73 CD90 CD105 CD166 CD14 CD31 CD34 CD45

Surface Antigen

PercentCellsPositive

ATCC Primay Cell Solutions adipose-derivedmesenchymal stem cells were taken fromliquid nitrogen and cultures initiated. Asample for analysis by low cytomety wastaken when the culture was initiated andthen after 48-hours of growth. The cells musttest positive for CD29, CD44, CD73, CD90,CD105, and CD166 (greater than 95% of thecell population expresses these markers bylow cytomety). The cells must test negativefor CD14, CD31, CD34, and CD45 (less than2% of cell population expresses thesemarkers by low cytomety).

Fresh from Cyo 48 Hours Growth






Materials Needed


Mesenchymal Stem Cells Growth Kit Options Basal Medium

Adipose-Derived Mesenchymal Stem Cells,Normal, Human (ATCC PCS-500-011)

Mesenchymal Stem CellGrowth Kit-Low Serum(ATCCPCS-500-040)

Mesenchymal Stem CellBasal Medium (ATCC PCS-500-030)

Umbilical Cord-Derived Mesenchymal StemCells; Normal, Human (ATCC PCS-500-010)





Prepaation of Complete Growth Media1. Obtain one Mesenchymal Stem Cell Growth KitLow Serum from the freezer; make sure that the

caps of all components are tight.

2. Thaw the components of the growth kit just prior to adding them to the basal medium.

3. Obtain one bottle of Mesenchymal Stem Cell Basal Medium from cold stoage.


5. Using aseptic technique and working in a laminar low hood or biosafety cabinet, tansfer theindicated volume of each growth kit component, as indicated in the following table, to the bottleof basal medium using a sepaate sterile pipette for each tansfer.

Mesenchymal Stem Cell Growth KitLow Serum (ATCC PCS-500-040)


MSC Supplement 10 mL

2% FBS5 ng/mL rh FGF basic5 ng/mL rh FGF acidic5 ng/mL rh EGF

L-Alanyl-L-Glutamine 6 mL 2.4 mM


7. Complete growth media should be stored in the dark at 2C to 8C (do not freeze). When storedunder these conditions, complete growth media is stable for two weeks.


1. Refer to the batch specific information provided on the last page of the product informationsheet for the total number of viable cells recovered from each lot of ATCC Human MesenchymalStem Cells.


3. Prepare the desired combination of lasks. Add 5 mL of complete growth medium per 25 cm2of




page 27 [email protected] [email protected] Phone800.638.6597page 27

suface area. Place the lasks in a 37C, 5% CO2, humidified incubator and allow the media to pre-


4. While the culture lasks equilibate, remove one vial of ATCC Human Mesenchymal Stem Cells fromstoage and thaw the cells by gentle agitation in a 37C water bath. To reduce the possibility ofcontamination, keep the O-ring and cap out of the water. Thawing should be apid (approximately

1 to 2 minutes).5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by

dipping in or spaying with 70% ethanol. All opeations from this point onward should be carriedout under strict aseptic conditions.


7. Tansfer 1.0 ml of the cell suspension to each of the pre-equilibated culture lasks prepared insteps 1 to 3 of Handing Procedure for Frozen Cels and Initiation of Culture.Pipette seveal times,

then cap and gently rock each lask to evenly distribute the cells.8. Place the seeded culture lasks in the incubator at 37C, 5% CO

2atmosphere. Incubate for at least


Maintenance




4. Add 5 mL of fresh, pre-warmed complete growth medium per 25cm2of suface area and return the lasks to the incubator.

5. After 24 to 48 hours, view each lask under the microscope todetermine percent cellular conluence. If not ready to passage,repeat steps 3 and 4 as described above. When cultures havereached approximately 70% to 80% conluence, and are activelyprolifeating (many mitotic figures are visible), it is time to

subculture.Subculture

1. Passage normal mesenchymal stem cells when the culture has reached approximately 70% to80% conluence.





6. Gently rock each lask to ensure complete coveage of the typsin-EDTA solution over the cells,

Note:Adipose- and umbilical cord-derived stem cells are contactinhibited. It is essential that thecells be subcultured BEFOREreaching conluence as post-conluent cells exhibit changes inmorphology, slower prolifeation,and reduced differentiation

capacity after passaging.


30/35www.atcc.org page 28www.atcc.org page 28

Human Mesenchymal Stem Cell Differentiation

and then aspiate the excess luid off of the monolayer.


8. When the majority of cells appear to have detached, quickly add an equal volume of the Typsin

Neutalizing Solution (ATCC PCS-999-004) to each lask. Gently pipette or swirl the culture toensure all of the typsin-EDTA solution has been neutalized.





13. Centrifuge the cells at 150 x g for 3 to 5 minutes.14. Aspiate neutalized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL

fresh, pre-warmed, complete growth medium.

15. Count the cells and seed new culture lasks at a density of 5,000 viable cells per cm2.



Adipose-derived Mesenchymal Stem Cell Differentiation Protocols

Adipocyte Differentiation

Materials NeededThe Adipocyte Differentiation Toolkit (ATCC PCS-500-050) contains medium and reagents designedto induce adipogenesis in actively prolifeating Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-500-011) with high efficiency, and suppot matuation of derived adipocytes during lipid accumulation.

Preparing Cells for Adipocyte Differentiation

1. Follow the instructions for the growth of Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-500-011). It is recommended that the cells not be passaged more than four (4) times beforeinitiating adipocyte differentiation.

2. When cells are 70-80% conluent, passage them into a tissue culture plate at a density of 18,000

cells/cm2. Adjust the number of cells and volume of media according to the tissue culture plateused.

Example: For a 6-well tissue culture plate with a suface area of 9.5 cm 2/well, add a total of171,000 viable cells to each well containing 2 mL of Mesenchymal Stem Cell Basal Medium (ATCCPCS-500-030) supplemented with Mesenchymal Stem Cell Growth KitLow Serum (ATCC PCS-500-040) components.

3. Gently rock the plate back and foth and side to side to evenly distribute cells before incubation.Do not swirl.

4. Incubate the cells at 37C with 5% CO2for 48 hours before initiating adipocyte differentiation.

Adipocyte Differentiation Media Prepaation

The adipocyte differentiation process requires twosepaate media prepaations: one for initiation and


31/[email protected] Phone800.638.6597page 29page 29 [email protected] [email protected] Phone800.638.6597page 29


one for maintenance. Stock solutions of these media can be prepared in tandem in advance as follows:

1. Thaw all three components of the differentiation kit and warm to37C in a water bath.

2. Decontaminate the external sufaces of all three kit componentsby spaying them with 70% ethanol.

3. Using aseptic technique and working in a laminar low hood orbiosafety cabinet:

a. Tansfer 15 mL of Adipocyte Basal Medium and 1 mL of AD Supplement to a sterile 50 mLconical tube, using a sepaate sterile pipette for each tansfer. This is your working stock ofAdipocyte Differentiation Initiation Mediumused during the first 96 hours of differentiation.

b. Add 5 mL of ADM Supplement to the remaining 85 mL of Adipocyte Basal Medium. This isyour working stock of Adipocyte Differentiation Maintenance Medium.

4. Tightly cap the each container of media and swirl the contents gently to assure a homogeneoussolution. Do not shake forcefully to avoid foaming. Label and date the bottle.

5. Each container of differentiation medium should be stored in the dark at 2C to 8C (do notfreeze). When stored under these conditions, the differentiation media is stable for up to threeweeks.

Adipocyte Differentiation Procedure

A. Initiation Phase

1. After incubating the prepared Adipose-Derived Mesenchymal Stem Cells for (as describedabove), carefully aspiate the media from the wells.

2. Immediately rinse the cells once by adding 2 mL of room-tempeature D-PBS (ATCC 30-2200) to each well, then carefully aspiate the PBS from the wells.

3. Add 2 mL of pre-warmed (37C) Adipocyte DifferentiationInitiation Medium to each well to begin the adipocytedifferentiation process.

4. Incubate the cells at 37C with 5% CO2for 48 hours.

5. Feed the cells by carefully removing half the volume of media(1 mL) from each well and adding another 2 mL of pre-warmed(37C) Adipocyte Differentiation Initiation Medium to eachwell.

Impotant:DO NOT TILT plate during aspiation. It is impotant that the cell monolayer is not

exposed to air during this and subsequent steps to ensure that developing lipid vesicles donot burst.

B. Maintenance Phase

6. Incubate the cells at 37C with 5% CO2for 48 hours.

7. Carefully remove 2 mL of media from each well (leaving1 mL) and replace with 2 mL of pre-warmed (37C)Adipocyte Differentiation Maintenance Medium in eachwell.

Impotant: DO NOT TILT plate during aspiation. It is

impotant that the cell monolayer is not exposed toair during this and subsequent steps to ensure that

Adipocytes differentiated from Adipose-derived Mesenchymal Stem Cells (PCS-500-011) and stained with Oil Red O todetect the formation of lipid droplets.

Note:It is recommended that youtansfer the required volume ofmedia to a sterile tube for pre-warming prior to each feedingather than repeatedly re-warmingthe entire working stock.

Note:It may be necessay to shake theAD Supplement and the ADMSupplement upon warming tohelp re-dissolve any componentsthat may have precipitated out ofsolution upon freezing.


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developing lipid vesicles do not burst.8. Repeat Steps 6 and 7 evey 3-4 days for another 11 days until adipocytes reach full maturity.

(Full maturity will be reached 15 days after the beginning of initiation phase, or 17 days frominitial plating of cells.)

9. Cells can be used at any phase of adipocyte differentiation as predicated upon experimental

design. To confirm lipid accumulation, cells can be fixed and stained with Oil Red O.

Additional differentiation protocols, including

Chondrocyte and Osteocyte differentiationof Adipose-derived Mesenchymal Stem Cells,are available online at www.atcc.org

ATCC Adipose-Derived MesenchymalStem Cells were expanded in CompleteGrowth Medium and then induced todifferentiate to chondrocytes usingthe Chondrocyte Differentiation Tool.Passage 3 cells, day 21 followingdifferentiation (100X magnification),stained with Alcian Blue.



REFERENCES1. Stein GS, et. al. Human Stem Cell Technology and Biology: A Research Guide and Laboatoy

Manual. Wiley & Sons, Hoboken, New Jersey, 2011.

2. Zuk PA, et al. Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Theapies.

Tissue Eng. 7(2):211-228), 2001.3. Zuk PA, et al. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell. 13:4279-

4295, 2002.

4. Ogawa R. The Impotance of Adipose-Derived Stem Cell and Vascularized Tissue Regeneation inthe Field of Tissue Tansplantation. Cur. Stem Cell Res. 1:13-20, 2006.

5. Shaeler A & Buechler C. Concise review: adipose tissue-derived stromal cellsbasic and clinicalapplications for novel cell-based theapies. Stem Cells 25:818-827, 2007.

6. Kang SK, et al. Neurogenesis of Rhesus adipose stromal cells. J. Cell Sci. 117:4289-4299, 2004.

7. Safford KM, et al. Stem cell therepy for neurologic disorders: Theapeutic potential of adipose-

derived stem cells. Current Drug Targets 6:57-62, 2005.8. Safford KM, et al. Chaacterization of neuronal/glial differentiation of murine adipose-derived

adult stromal cells. Exp. Neurol. 187:319-328, 2004.

9. Kingham PJ, et al. Adipose-derived stem cells differentiate into a Schwann cell phenotype andpromote neurite outgrowth in vitro. Exp. Neurol. 207:267-274, 2007.

10. Xu, et al. Myelin-forming ability of Schwann cell-like cells induced from at adipose-derived stemcells in vitro

ATCC Primary Cell Culture Guide

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