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466 M II(}CHONDRTAL DISEASES AND ACINC I40l l40l MYOBLAST CULTURES '

Epidermal growlh fad6. IResearch 40052, Bedfor4Q water atrd fre€ze iD 1-l

5.1H11 mouse monodotrdnot commercially ar€ildHospital, Lordoo, UK tto us); the urdituted httblasts

Fluorescein{oD.iugated F(dglobulin c 0gG) (0m1311009); dilure 1 :1m il

Cell Culture Media

Suppleme ed growrh Eo(GLI_TTAMAX, GIBCO)1 liter of liquid: 5m Eg tliml conceDtmtion), 5mtrcorcentrution), 1m dg intion), 10 n of stock sohml, fnal concentration), ang/n , final cotrceDtratidfilter, then add 15% feral (ar 56") aDd genramicin (c

Fusion medium: DulbecEob2% hors€ serum (beat itra

Freeziry mediumt Ham's Fl(sufoxide (DMSO)

Washing mediuE: DMEM oand 2.5 pglmt gedamhi!

N.nes on Culture Mdb- Hagrowth ot human musd€ sat€nitEtHe started our work thb 'Erlilavailable commercially ftm Oanent ir fetuin is litely a tra€ cbe separated from purifed Ped.dnart also contdbutes to the €&amuch higher than physiological.cloned in serum-free MCDB li!above; however, growlh is sup€rj$rith 15E FCS.4

52. Nic, D. Jelirck, dd R- C. Ham, B

mutations.2 One can also detemine the threshold for phenot!?ic expressi onofpathogenic miDNA mutanls, as the degrce of heteroplasmy in nrdividualmyotubes can be manipulated by seeding cultures with myoblasts containingdifferent pmportions of mulanl and wild type mtDNAs. Second, becausesalellite sels are dormant most of thc time, it is likely that the turnoverof mitochondria and mtDNA is extremely slow as comparcd to that indilferentiated muscle fibers. Thus, the rclative proportions of mutatrt andwild type mtDNAs in the satelite cell population likely reitects thc degreeof heteroplasmy that existcd in fte myoblast precursor population duringembryological developmcnt. A compa sotr with adulr muscle can provideinformation on the segregation ofmlDNAs subsequent to terminal differen-

In this chapter we detail the procedures we have used tor (i) directcloning oI primary human muscle saiellite cells, (ii) immunolluorescentstaining of myoblasls for fluorcscerce-activated cell sorting (FACS), and(iii) analysis ol mitochondrial translation products in myoblasts and myo-tubes. The culture methods wc have used ar:e based on the pioneedng workcarried out in tle laboratories of Blau and Ham.3!

Materials

Solutions and Reagentr for Cell Cultwe

Solution A: l0 mM glucose, 30 mM HEPES. i30 mM NaCl, 3.0 mMKCl. 0.0033 mM phenol rcd; mdte up in Milli-O water, adjust topH 7.6 with NaOH, and sterile filter

Solurion Aplus 0.05E (w/v) crystalline trypsin (GIBCO, crand lsland.NY) atrd 0.027, (w/v) EDTA;sterile liltcr and storc frczen ir aliquotsal 20'for up to 1 month

Phosphate buffered saline, Mg'z*- and Ca'?rftee (PBS): 8 g NaCI,0.2 g KC],1.15 g Na,H POa,0.2 g KH,POa;dissolve in 1 litcr Milli Qwaier and autoclave

PBS plus 0.05E (w/v) crystalline trypsin (GIBCO) and 0.02Ea (wlv)EDTA; slerile filter and sto.e at 4' fbr 1 week

PBS plus 0.57. bovine serum albumin (BSA); sterile 6lter atrd store

Dexamethasone (Sigma, Sr. Louis, MO;0.039 mg/ml); dissolve in MilliQ water and lieeze at 20'in 10-n ahquots

rL. Bodet, C. Karpati, and E. A. Shoubidge,.4u J. Hun. Cenet.5l.ll87 (79q./).3H. M. Blau a C. Webster, Ptu.. Natl. A.ad ScL U.S.A. 7& 5623 (1981).a R. G. Ham, J. A S1. C1dir, C. Websler, and H. M. Blau, 1" ytl, Cell. Dev. Bio1.A,831llgaa).

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468 I40l 14OI MYOBLAST CLLTI-TIES

Direct Claning of Hunan Muscl( Sate ite Ce s

1. Obtain skeletal 'nuscle

(-50 100 rng) .rseprically from a biopsyspccimen and place in a 15-ml sterile tube containing l0 ml solution A.Hanks'balanced salt solution or otter sterile saline solutions can also beused. Place on icc immediately and store at 4'. The musclc can be kept forup to 5 days without significant loss oI proliferative capacity ofthc satellitecels, alihough s'e llsually proceed with the cuiture within 1 or 2 days.

2. Tra$fer the muscle sample to a preweighed 60-mm tissuc cultutedish and rinse with 5 ml solution A. Cul the biopsy into small picces(1 rnm'z) with a pair of small, sterile scissors in 5 m], solution A plustry?sin and EDTA. Transfer tlre minced tissue to a meaton (Milville,NJ) trypsinizing ffask cortainiry a magretic stirring bar- RiDse the culturedish rwice more with 5 rnl solulion A plus lr'?sin and EDTA, each timetr rsferring tl}e tissue fiagmenls and solution to the trypsinizing ilask.

3. Stir the tissue pieces vigorously for 15-20 min on a stirring pialeat 31".'|\\is is accomplishedb) plachg a \rater balh (a qrrex drsh is suitab\Oon top of tlle magnetic slirrer in a laminar flow hood. Allow ttre tissuedcbris to seltle for abour 1 min and decant the solution containing thedissocialed calls into a 50-ml centrifuge lube coniaidng an equai volume(i5 ml) of washing medium.

4. Rcpcat step 3 two more times (threc limes in a[), each time adding15 ml solution A plus typsin and EDTA to the remaining rissue in thetl,psinizing flask.

5. Spin the cclls at approximately 500 g (-1450 rpm on a BeckmanGP benchtop centrifuge, Mississauga, Ont.) for 10 min and discard thesupernatani. Resuspend thc pellel in 2 nrl SGM. Pool the ccll suspensionsfrom each dissociation.

6. The suspeffion of dissociatcd ceils will contair many eryrhrocytesand small tissue fragments. To clone myoblasts directly it is necessary 10estimate thc number of satellite cells that wefe preseDt ir the originalbiopsy malerial because they cannot be easily counted in the suspeflsion.We have used an cslimate of 500 satelite cells/mg we! weight of skeletalmuscle. Accordingly. dilutc the cell suspension and plate in 0.2 ml growthmedium in 96-we11 plates (Costar, Cambddge, MA). Because the numberof satellite cells in the sample is a rough estimate, we routirely dilute thcoriginal sample to theoretically 2.5, 5, and 10 cels/lnl, which would give0.5, 1, or 2 cells per well on a 96-well plate. We sei up duplicate plates ateach dilution. which in practice usually allows us to isolate 40-50 clones.It is also possiblc 10 plate out lhe cells at this stagc at very low dilutionand to rescre colonies subsequertly using cloning rings: however, we have

found cloning by limiting diluriclure. Plate lhe remaining cdkThis produces a bulk oitrecontaminating fibroblasrs or rra humidified CO, incubarq d

7. Single ce s shouldb.riphase contrast micrccope. |l ito score wels containing dd.r\Wells that were seed€d rirh !because more than orre f(rshouid be changed ever-y r€€llwo-thirds conflueot rra&Aspirate the m€diuo aod dPlace the plate at 3f f6 a qUsing a Pipetman. disp€re riaml SGM.

8. When rhe clones ar€ €laspimte the SGM, wash sirh Oand EDTA to each well. A+ithc incubaror for a couple of rdispersewirh a pipelle- At thb!of the clones. We usuallv trdEculture dish in 5 ml SGM io.to a 24 wel plate in l5 ml SG

9. Myobtasts.'arely fr|s€ ilactors; therefore. it is aeG:ssr(fusion) rnedium. cros rL cdto confluence and replace SGXof multinucleate myotutr€s 2-3micmgraph of a clonal 6_r,obtthe same culture .|8 hr aier srilarge differetrces ir lhe deg€€from the salne biop6y. and Eat this stage for use in laler qand stain the nuclei of these cafusion is desired. In our elperany evidcnce of cell fusion- as

10. The final step involv€sthe cells have reached aboulthey can be frozen. (It is alsonumber ofcells at early passag€about 1 nl] PBS plus trpsin aDplace the cells in the incubaro

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MYOBLAST CULTLTES

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472 MITOCEONDRIAL DISEASES AND AGINI] I40l

reaction witll 5 ml GM, then rinsc lhe plate and dispene the cells with anaddilional 5 ml SGM. Spin the cells at approximately 500 g and gcnuysusp€nd in 1.5 n of cold freezing medium. Transfer to freezing vials (Nunc)and place in a foam rack at 70'ovemighl. AJter 24 hr the cells can betransferred to a liquid nitrogen freezer. To thaw myoblasts, quickly warmthe ftozen vial in a 37" water batll until tlle contents have alinost thawed.Immerse tlle vial in 7070 (v/v) ethanol to steilize and uansfer the entireconlents to a tissue culture plate containing SGM. AAer tlc cclls have plateddown (3 4 hI or overdght) changc t}le medium to eliminate the DMSO.

nuorcscence-Acttuated Ce Sorting of Euman Sateltile Calb

It is sometimcs usefr to have a large populalion of pure skeletalmyoblasts from a biopsy sample at ar early passagc. In this case purillingmyoblasts using a FACS machine is a much more eflicicnt altemative tocloning.This technique isbasedon the use of a mouse monocional aDtibody(5.1H11) raised against human myotubes and subsequently idenrlfied asanti-N CAM (neuml cel adhesion molecule).6.7 N-CAM is present on rhesurtace of primary humar satelite cells, bulnot on skeletal libroblasts thalcontaminate pdmary muscle cel sultures. Thc method we have used issimilar to that describcd by Webster

"tdl6 Below we descdbe our method

for immunostaining myoblasts with fluorescein for sorting. Alt proceduresale pe{ormed sing sterjle technique.

1. Expand the cells in the bulk myoblast culture until lhere are 4-5 x106 cels. Although fewer cclls can be used, it is better to starl wilh severalmillion because there are unavoidable losses during the sofiing procedure.Dissociale the cells in PBS plus trt?sin and EDTA ard resuspend in SCM.Centifuge at 4" for 10 min at 11,000 +m in a microcentrituge and discardt-he supematant.

2. Transfer the cells to a stedle, 1.5-ml Eppendort tube at 4 x 106 cellsper tube and suspend in 1.0 nn PBS plus 0.5E BSA. Centrifuge 1 min(setting 4 on a Fisher microcentrifuge) at room temperarurc and discardthe supematant.

3. Suspend the cells as above and add 100 pI anti-NCAM antibody(undiluted supernalant ftom hybridoma cells). Mix ard incubate or ice inthe dark for 45 min, mixiDg every 15 min.

4. Wash the cels 3 times wjtr PBS plus 0.5E BSA. Cenlriluge for 1min at setting 4 on the microcenlrifuge between each wash.

6 C. websler, G. K. Pavlnth, D. R. Parks, F. S. walsh, and H . M. l3la\ Lxp. CeU Rs. O4,252 (1988).

7 F. S. Walsh,,4dr. Ev. M./ Rtrr 280, 41 (1990).

t4ol MYOBI-AST CLATURES /

5. Aftcr the lasr vrash srqrBSA ard add 100 rrl FTTC (dibddrk for 45 mirl.

6. Wash the ce s 3 timesridat a concentration of ,l-5 x td

7. The details of the actlal s{available and are b€st dealt dlconsultation with an ef,Frleused a single laser machioe (Btures of myoblasls atrd skel€olparameters which alloxr soditrgtulalions. The negative celh (*at the same time and ||sed to €!

AnalJsis of Mitochondial Tr

Most repofted mitochoadripoirt mufations or large-scale dcodirg and tRNA genes)- Borhaffect the translation of mtDMof the I 3 proteins encoded in mdmyoblasts or myotubes {,irh Fan inhibitor of cltoplasmic Earbe tesred by adding chloraqt rlation, to the labeling mixture,1developed by Attardi and Giq

Sotunons fot LabelinS Tr@a

DMEM wirhout methi@bEmetine (Sigma), stoci sd

ter stedlizeChloramphe col(Sigma),

and lilrer sterilizel35s]methionine (sp€ci6c {

1. Grcw myoblasts to sutrcormyotubes, switch the mediurD aoconltuent and label 48-72 hr l,Nt

! G. Attardi and E. Ching. tl s *.i€*i

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474 I40l t40l MYOBLAST (-LTL?ES

2. Wash lhe cells twice with PBS and incubate in 2 ml DMEM withourmethionine for 30 min. Add 100 &l enetine (6na1 concenrrarior 100,uglml) ard incubare lor afurther 5 min- Add chloramphenicol (fina1concentration 100 i,g/ml) plus emetine to a parallel, negative conlrol plare. Allincubations are donc in a humidilied CO, incubator ai 37'.

3. Add 200-100 pci [3is]methionine to each plate and incubale for upto t hr. Aspirate tlle medium, replace with rcgular DMEM, and incubarefor 10 min-

4. Wash the cells gently three times ir PBS, trypsinize, and pellet thccells in excess PBS on a benchtop centdfuge at approximarely 1000 g arroomtemperature. Note that a brief trypsinization period pen ts isolationof essentially pure myotubes, free of any mfused mononuclear myoblasts.which arc more tightly allached to tissue cullure plastic.

5. Suspend thecellsin 200rtlPBS and detcrmine theprotein concentra-tion. Pellct cels (30-50 ,ug prolein) at top speed in a microcentrifuge andsuspend in 10 rtl water plus 10 pl gcl loading solution [86 mMTris-HC], pH6;7, 15E g1ycercl,'7qa sodium dodecyl sulfate (SDS). 67. mercaptoerhanol,0.0058 bromphenol bluel. Sonicate the suspension for 3 sec (609o on a

Vibracell sonicator, Sod6 afttop speed in a microcenrrifuge

6. Run thc samples oD a liprocessed lbr fluorogaph\. for ephy lbr analysis on a phospbodevice.r Figure 2 shows an analylrn nyotubes from a conEol atr(ai position 8344 in tRl\IAL).-ro

Addiuonal Comments

The proliferative potenriat (that one can obtain abour 60 ,(1.1 x 1013 cells). bur only 20 (It is, howevcr, impo.tatrr to norcas thc cells approach senesceftexpenment, the cells have to bwise tolimit the numberofceldIr practice tbis can be difficulr !from the same patienr can vawcompetence. It is importanr roto pefibrrn the experiments aftrThe fusion medi m we have userbeyoid ?2 hr. Long telm surviaddition of insutin (10 pglrDl)- lthe lusion medium.11

Acknowledgments

Thn rescarch was supported bt g.ethc Musculd Dlstophy AssiandK. McDonlld lor tecbnical cisla@.

' \J. K.laennnlj, Nalute (Lddo.) Z,'"A. Chonyn, c. Meoia, N. Br€ti.. s.

1! 226 (1991).I J. A. St. Cian, S. D. Mete.Deddeg

ND5.

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