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Methods in Molecular Biology, Vol.005 - Animal Cell Culture 1st Edition

Methods in Molecular Biology, Vol.005 - Animal Cell Culture 1st Edition

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Chapter 1
Basic Cell Culture
Jeffrey W. Pollard
1. Introduction
This article will describe the basic techniques required for successfulcell culture. It will also act to introduce some of the other chapters in thisvolume. It is not intended, as this volume is not, to describe the establish-ment of a tissue culture laboratory, nor to provide a historical or theoreticalsurvey of cell culture. There are several books that adequately cover theseareas, including the now somewhat dated but still valuable volume by Paul(I), the mult’ r-authored Methods in Enzymology volume edited by Jakobyand Pastan (Z), and the new edition of Freshney (3). Instead, this chapter’sfocus will be on the techniques for establishing primary rodent cell culturesfrom embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of geneticallypure strains. The cells described are all derived from Chinese hamsterssince, to date, these cells, have proved to be the most useful for somatic cellgenetics (4,5). The techniques, however, are generally applicable to mostfibroblastic cell types.I will only discuss growing fibroblastic cells in semidefined media. Avery detailed consideration of serum-free culture and the maintenance ofepithelial cells can be found in Chapter 21. Methods for culturing manyother nonfibroblastic cell types are described in Chapters 2 through 31.1
 
2 Pollard
2. Materials
1. Alpha minimum essential medium (a-MEM): for economy, we buyprepared medium as powder. A 44 g quantity of sodium bicarbonateis added, the powder is made up to 20 L in deionized distilled water,the pH adjusted to 7.4, and the media sterile-filtered through a 0.22 wfilter using a pressure vessel coupled to a filtration apparatus anddriven by a pressurized 95:5% CO, gas mix. This gas mix maintainspH upon storage. The 500 mL bottles are stored at 4OC n the dark untiluse (seeNotes 1 and 2).2. Growth medium: a-MEM plus 15 or 7.5% (v/v) fetal calf serum. Thisis made up as required and stored at 4OC.3. Ca2+Mg2+-free Phosphate Buffered Saline (Dulbecco, PBS): NaCl, 8 g/L; KCl, 0.2 g/L; KH,PO, 0.2 g /L; Na,HPO,
l
7HZ0, 2.31 g /L pH 7.2.4. PBS citrate: PBS + sodium citrate at 5.88 g/L.5. Trypsin: One vial of lyophilized Difco Bacto-trypsin in 400 mL of PBScitrate (0.125% trypsin) or 10 x this concentration for the isolation ofembryonic fibroblasts (seeNote 3).6. Counting fluid; PBS + 0.2% (v/v) FCS.7. Formalin fixative: 10% (v/v) commercial formaldehyde (comes as a40% v/v solution).8. Methylene blue stain: 0.1% (w/v> methylene blue in distilled waterfiltered through a Whatman No. 1 filter.9. Trypan blue: 0.5% (w/v) in PBS.10. Colcemid: 10 pg/mL, stored at 4OC.11. Karyotype fix: Methanol: acetic acid (3:l) made up on the day of useand kept on ice in a tightly stoppered bottle.12. Giemsa stain: Use commercial Giemsa concentrate diluted 3:47 partsin commercial Gurr’s buffer (one tablet to 1 L distilled water). Alter-natively, 10 mM potassium phosphate, pH 6.8, can be used as thebuffer. The diluted stain is only stable for 2-3 mo.
3. Methods3.1.
Establishment of Primary ChineseFibroblast CuZtures
3.1.1. Embryo Culture
1. Kill a 10-12-d pregnant Chinese hamster with ether.2. Wash the animal in tap water and then with 70% ethanol.
 
Basic Cell Culture
33.4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.Make a surgical incision on the dorsal side to expose the uterus usingsterile instruments (these can be dipped in ethanol and flamed tomaintain sterility during the operation).Remove the uterus
in
toto, and transfer it to a sterile Petri dish. Dissectout the embryos, and place them in a new sterile Petri dish (seeNote4).Mince the embryos very finely, and while still in the Petri dish, washthe pieces with 5 mL of 0.125% Bacto-trypsin at 37OC.Tilt the Petri dish so that embryo pieces go to the side. Remove thepieces into a 50 mL centrifuge tube using a wide-bore pipet.Add 40 mL of fresh 1.25% Bacto-trypsin, and incubate at 37OC or 5min.Regain the embryo pieces by centrifugation at 100s for 3.5 min, anddiscard the supernatant.Resuspend the pieces in 40 mL of fresh 0.125% Bacto-trypsin, and in-cubate at 37OC or 25 min (this can be performed in a roller apparatus).Neutralize the trypsin with 4 mL of FCS.Deposit the supernatant through a 100 pm sterile mesh into anothercentrifuge tube.Centrifuge the supernatant for 5 min at 3008 at room temperature.Resuspend the pellet in 10 mL a-MEM plus 15% FCS, and count thecells in a hemocytometer at about l/100 dilution.Lay down 1.5 x 1O’cells n 40 mL of a-MEM plus 15% FCS nto a 75 cm2,and place it in a 37OC issue culture incubator.The next day, replace the medium with an equal volume of a-MEMplus 15% FCS.Forty-eight to72 h later, the monolayer should be confluent, and at thispoint, the cells are ready for subculture. This is performed byincubat-ing the monolayer with 4.5 mL of 0.125% Bacto-trypsin at 37OCuntilthe cells detach. Cell detachment can be visualized either by observ-ing the cell monolayer in oblique light or directly under the micro-scope. When the cells have detached (-go%), add 0.5 mL FCS (lo%),pipet up and down five times, and transfer contents to a 15 mLcentrifuge tube.Centrifuge the cells at 3008 for 3.5 min at room temperature.Remove the supernatant, resuspend the cell pellet in 5 mL a-MEMplus 15% FCS, and determine the cell concentration.Resuspend the cells at 4 x 106/vial in a-MEM plus 15% FCS plus 10%(v/v) sterile DMSO, and freeze at -120 or -176OC.The cells will remainviable for several years.

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