Improved Media for Normal Human Muscle Satellite Cells: Serum-Free Clonal Growth andEnhanced Growth with Low SerumAuthor(s): Richard G. Ham and Helen M. BlauSource: In Vitro Cellular & Developmental Biology, Vol. 24, No. 8 (Aug., 1988), pp. 833-844Published by: Society for In Vitro BiologyStable URL: http://www.jstor.org/stable/4296304Accessed: 17/08/2010 15:59

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IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Volume 24, Number 8, August 1988 ? 1988 Tissue Culture Association, Inc.

IMPROVED MEDIA FOR NORMAL HUMAN MUSCLE SATELLITE CELLS: SERUM-FREE CLONAL GROWTH AND ENHANCED

GROWTH WITH LOW SERUM

RICHARD G. HAM', JUDY A. ST. CLAIR, CECELIA WEBSTER2, AND HELEN M. BLAU

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309 (R. G. H., J. A. S.) and Department of Pharmacology, Stanford University

School of Medicine, Stanford, California 94305 (C. W., H. M. B.)

(Accepted 17 May 1988; editor David W. Barnes)

SUMMARY

We have developed a serum-free medium for clonal growth of normal human muscle satellite cells

(HMSC). It consists of an optimized nutrient medium, MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum

albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as

the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further

improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal

growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and

monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the

capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells.

Key words: MCDB 120; EGF; FGF; fetuin; albumin; pituitary extract.

INTRODUCTION

Clonal growth of mononucleate cells from embryonic chicken skeletal muscle and their fusion and differentia- tion to form multinucleate contractile myotubes was achieved relatively early in the history of modern cell culture [reviewed by Konigsberg in ref. (30)]. Growth of human skeletal muscle cells was also achieved quite early (26). However, despite many years of studies on growth and differentiation of skeletal muscle cells in vitro, relatively little attention has been given to the develop- ment of culture media optimized specifically for growth of muscle cells from humans or other species.

Nutrient medium F10 (20) was used with serum and chicken embryo extract to grow chicken embyro muscle cells over 20 years ago (27) and human muscle cells a few

years later (26). Despite its lack of modern features, F10 plus serum and embryo extract has continued to be

widely used for muscle cell culture, including establish-

ment of clonal cultures from normal and diseased human muscle cells (7,8). The most frequent alternative is Dulbecco's modified Eagle's medium (DME), which was

developed in the same general time period and has many of the same limitations. Other nutrient media that have been used for muscle cell cultures, sometimes in combination with DME, include M199 (2,30), MEM plus nonessential amino acids, pyruvate and additional vitamins (37), RPMI 1640 (28), F12 (40), F14 (3,44), MCDB 104 (1), and MCDB 201 (14).

We have undertaken a systematic study of the growth requirements of normal human muscle satellite cells (HMSC). This study resulted in the development of a new nutrient medium, MCDB 120, optimized specifically for clonal growth of HMSC, as described in this paper. We have also achieved serum-free clonal growth of HMSC by adding a set of five defined and semi-defined supple- ments (SF) to the optimized nutrient medium. There have been previous reports of "defined" media for HMSC

(3,11,45), but those media have all been more favorable for differentiation than for growth. In addition, they required inoculation of dense populations of cells into a

serum-containing medium, followed by medium change to the serum-free formulation, whereas we are able to

' To whom requests for reprints should be addressed at M.C.D. Biology, Campus Box 347, University of Colorado, Boulder, CO 80309.

2 Current address: Department of Biochemistry, University of California, Riverside, Riverside, California 92521.

833

834 HAM ET AL.

obtain good clonal growth from a small number of

trypsinized cells inoculated directly into the final serum-free medium.

We have also found that doubly-supplemented media containing both SF and 5% dialyzed fetal bovine serum support far better growth than media with either alone. Clonal growth in doubly-supplemented MCDB 120 is far better than with any amount of serum and embryo extract, and monolayer growth at least equals that previously obtained in conventional media with optimal levels of serum and embryo extract.

MATERIALS AND METHODS

Chemicals. All biochemicals were from Sigma (St. Louis, MO) and all inorganic salts were from Fisher Scientific (Pittsburgh, PA), except as indicated. Epider- mal growth factor (EGF) was from Collaborative Research (Waltham, MA). Fibroblast growth factor (FGF) was from

BRL (Gaithersburg, MD). Highly purified full length acidic FGF (prostatropin) was generously provided by Dr. Wallace McKeehan (W. Alton Jones Cell Science Center, Lake Placid, N.Y.). Fetuin prepared by the method of Spiro (42) was from GIBCO (Santa Clara, CA). Water for medium preparation was prepared by reverse osmosis, followed by passage through a Milli-Q ion

exchange system (Millipore, Bedford, MA). Media. Dulbecco's modified Eagle Medium (DME),

medium F10, and other "standard" media were pur- chased as dry powders from GIBCO. MCDB media were

prepared from the constituent chemicals in our labora- tory. MCDB 131M was prepared according to the protocol for MCDB 131 (29), except that the concentration of

magnesium sulfate was reduced to 1.0 mM. MCDB 120 (Table 1) is a new formulation developed for HMSC

during the course of these studies. It was prepared according to the protocol for MCDB 131 (29), with nutrient concentrations modified as described in Table 1.

TABLE 1

COMPOSITION OF MCDB 120 AND MCDB 131Ma

MCDB 120 MCDB 131Mb MCDB 120 MCDB 131Mb

M/L Mg/Ld M/Le M/Lc Mg/Ld M/L'

Amino Acids Thiamin - HCI 1.00E-5 3.373 L-Alanine 3.00E-5 2.67 Vitamin B12 1.00E-8 0.01355 L-Arginine - HCl 1.00E-3 210.67 3.00E-4 Other Organic Components L-Asparagine - H20 1.00E-4 15.01 Adenine 1.00E-6 0.1351 L-Aspartic Acid 1.00E-4 13.31 Choline Chloride 1.00E-4 13.96 L-Cysteine - HCI- H20 2.00E-4 35.13 D-Glucose 5.55E-3 1000.00 L-Glutamic Acid 3.00E-5 4.41 myo-Inositol 1.00E-4 18.016 4.00E-5 L-Glutamine 1.00E-2 1461.50 Putrescine- 2HCl 1.00E-9 0.0001611 Glycine 3.00E-5 2.25 Sodium Pyruvate 1.00E-3 110.04 L-Histidine - HCl H20 2.00E-4 41.93 Thymidine 1.00E-7 0.02422

L-Isoleucine 5.00E-4 65.58 Bulk Inorganic Salts L-Leucine 1.00E-3 131.17CaC2 - 2H20 1.60E-3 235.23

L-Lysinei- HCI 1.00E-3 181.65 KCI 4.00E-3 298.20

L-Methionine 2.00E-4 29.84 1.00MgSO4 7H20 1.00E-3 246.38

L-Phenylalanine 2.00E-4 33.04 NaCl 1.10E-1 6430.0 L-Proline 1.00E-4 11.51 L-Serine 3.00E-4 31.53 L-Threonine 3.00E-4 35.73 1.00E-4 Trace Elements L-Tryptophan 2.00E-5 4.08 CuSO4 -5H20 1.00E-8 0.002496 5.00E-9 L-Tyrosine 1.00E-4 18.12 FeSO4 -7H20 3.00E-6 0.8340 1.00E-6 L-Valine 1.00E-3 117.15 H2SeO3 3.00E-8 0.00387

Vitamins MnSO,4 5H20 1.00E-9 0.000241 d-Biotin 3.00E-8 0.00733 Na2SiO3 -9H20 1.00E-5 2.842 Folinic Acid (Ca (NHJ)6MO,0O24 4H20 3.00E-9 0.00371

salt) - 5H20 1.00E-6 0.602 NH4VO3 5.00E-9 0.000585 DL-Alpha-Lipoic Acid 1.00E-8 0.002063 NiCl2 -6H20 3.00E-10 0.0000713 Niacinamide 5.00E-5 6.11 ZnSO4- 7H20 3.00E-7 0.08625 1.00E-9 D-Pantothenic Acid 1.00E-4 23.82 5.00E-05 Buffers, Indicators

(Hemi-Ca salt) and Miscellaneous Pyridoxine - HCl 1.00E-5 2.056 Phenol-Red (Na salt) 3.30E-6 1.242 3.30E-5 Riboflavin 1.00E-8 0.003764 NaHCO3" 1.40E-2 1176.0

"Medium MCDB 120 and medium MCDB 131M are both prepared according to the protocol for MCDB 131, with appropriate changes in con- centrations as indicated in this table (29).

bConcentrations in MCDB 131M are listed only when they differ from those in MCDB 120. MCDB 131M is MCDB 131 (29) with magnesium sulfate reduced to 1.0 mM.

cMolar concentrations are expressed in computer style exponential notation. Thus, 3.00E-5 means 3.00 X 10-' moles per liter. dThese media are defined in terms of molar concentrations to three significant figures. Weight concentrations are expressed wtih sufficient

precision to yield accurate molar concentrations when divided by the molecular weights of the components. "To insure a correct final pH with 5% carbon dioxide incubation, the pH of MCDB 120 should be adjusted to pH 7.4 in air at room temperature

prior to addition of the sodium bicarbonate. The final osmolarity of the completed medium (with bicarbonate) should be 270 ? 5 mOsm/kg.

HUMAN MUSCLE CELL SERUM-FREE MEDIUM 835

Saline solution A (41) has the following composition: glucose, 10 mM; KC1, 3.0 mM; NaC1, 130 mM; Na2HPO4- 7H20, 1.0 mM; phenol red, 0.0033 mM; Hepes, 30 mM; and NaOH as needed to adjust final pH to 7.6.

Supplements. Fetal bovine serum (FBS) and horse serum were obtained from Hazleton Research Products (Lenexa, KS). Human serum was from Irvine Scientific (Santa Ana, CA). Dialyzed sera were prepared as follows: A buffer consisting of 2.0 M Tris, 0.6 M citrate, and 0.4 M borate at pH 7.0 was diluted 1:40 into the serum (usually 25 ml of buffer added to 975 ml of serum) and the mixture was stirred for 30 min on ice. The buffered serum was then transferred to tightly clamped dialysis bags and

dialyzed at 40 C against 100 volumes of deionized water for 3 days, with daily changes of water. This was followed

by lyophilization and storage of the dialyzed solids at -200 C. Convenient amounts were periodically dissolved in saline solution A at 50 mg/ml (total weighted solids), adjusted to pH 7.6, sterilized by membrane filtration (0.2 p, detergent-free), and stored frozen at - 200 C in small aliquots until needed.

Chicken embryo extract was purchased from GIBCO. Bovine extract (BPE) was prepared in our laboratory as

previously described (24,25). Full strength BPE contains

approximately 14 mg/ml protein (determined by the

Lowry method with serum albumin as a standard). Dialyzed BPE (dBPE) was prepared by dialysis against deionized water for 3 days (100 volumes, with daily changes), followed by centrifugation at 27 000 Xg for 15 min, and sterilization by membrane filtration (0.2 y, detergent-free).

Early studies were done in media GM-1 (F10 supplemented with 15% FBS and 0.5% chicken embryo extract) and GM-2 (F10 supplemented with 15% horse serum and 0.5% chicken embryo extract) as described by Blau and Webster (7). Most of the nutrient growth response titrations were done in medium MCDB 131M

supplemented with 5.0% dialyzed fetal bovine serum (dFBS) and 0.5% dialyzed BPE (dBPE). During the course of the research described in this paper, a serum-free supplement designated SF was developed (Table 2). In some cases, 30 ng/ml FGF was added to SF, as indicated in the text and tables. These supplements were used for serum-free growth both with MCDB 131M and with MCDB 120. The complete serum-free media are described in this paper by the name of the nutrient medium followed by "SF" (or "SF+FGF" when FGF was also added). Doubly-supplemented media containing both SF and 5.0% dFBS are identified by the name of the nutrient medium followed by DS (or DS+ FGF when FGF was also added).

Cells. All experiments were done with clonal cultures of HMSC prepared by direct cloning of satellite cells isolated from tissue samples (7). Initial experiments were done with clonal cultures shipped frozen from Dr. Blau's laboratory at Stanford to Dr. Ham's laboratory in Boulder. Recent experiments have generally been done with clonal cultures established in Boulder from muscle

biopsy discard tissue kindly provided by Dr. Hans Neville, University of Colorado Health Sciences Center,

TABLE 2

SERUM-FREE SUPPLEMENT SFa

Component Source b Final Concentration

Bovine serum albumin Sigma A-7888 0.50 mg/ml (Insulin RIA grade)

Dexamethasone Sigma D-1756 1.0E-6 M (0.39 ~g/ml)

Epidermal growth factor Collaborative 40001 10.0 ng/ml

Fetuin (Pederson) Sigma F-2379 0.50 mg/ml

Insulin (bovine) Sigma 1-5500 3.0E-5 M (180 Pg/ml)

aSupplement SF is used with MCDB 131M or MCDB 120 for serum-free clonal growth of HMSC. It is also used together with 5% dFBS in doubly-supplemented (DS) media. In some cases, fibroblast growth factor (BRL 6111-LA) is added at a final concentration of 30.0 ng/ml, as indicated in the text.

bCatalog numbers are provided to identify the components of SF precisely, since a number of other closely related products are also sold by the same suppliers.

Denver. In all cases, primary clonal cultures were grown up until there were enough cells for freezing (about 20

population doublings), and then stored frozen, as described by Blau and Webster (7).

Clonal growth assay. Ampules containing about one million frozen cells were thawed and distributed to two 25 cm' flasks containing either medium GM-2, or, in more recent experiments, MCDB 131M DS or MCDB 120 DS. The cultures were grown with medium changes as needed until they were semi-confluent, at which time individual flasks were used to prepare the inoculum for clonal

growth experiments or subcultured and again grown to

semi-confluency. Before the cellular inoculum was prepared, the test

media were prepared and 5 ml amounts were placed in triplicate 60 mm tissue culture petri dishes (Lux, Miles Laboratory, Naperville, IL). The dishes containing the media were then equilibrated in the cell culture incubator (370 C, 5% CO2 in air, saturated humidity) for at least one hour.

To prepare the inoculum, the medium was removed from the culture flask and the cells were rinsed with 2.0 ml of 0.05% (w/v) trypsin plus 0.02% (w/v) EDTA in saline solution A (pH 7.6) and then incubated in 2.0 ml of the same solution at room temperature until they were rounded up and beginning to release from the culture surface. The flask was then rapped sharply against the bench top to release the remaining cells and 2.0 ml of the medium into which they were to be inoculated was added. The suspension was centrifuged gently (1000 Xg for 3 min) and the supernatant was discarded.

The cells were then resuspended in complete growth medium (or growth medium minus the component being tested). A sample was counted in a hemocytometer, and dilutions were made as needed to obtain the desired number of cells in an inoculum of 50 pl. For the

836 HAM ET AL.

experiments described in this paper, the number of cells inoculated has varied from 1000 to 100 cells per dish, with a downward trend as growth has been improved. Most current growth-response experiments are done with 200 cells per petri dish, and an inoculum of 100 cells per dish is used for determination of colony forming efficiencies.

Immediately after the inoculum was added, the dishes were swirled gently to insure uniform distribution of the cells over the culture surface. The dishes were then incubated for 14 days without medium change. At the end of this period, the medium was discarded and the cells were fixed with methanol for 10 min. The dishes were then rinsed briefly with tap water. Two dishes from each set were then stained for 15 min with 0.1% crystal violet in water for evaluation of growth, and the third was stained for 15 min with Giemsa stain for evaluation of fusion.

Evaluation of growth. Total colony area per dish was determined with an Artek 880 colony counter that has been modified to detect areas with greater than a preset minimum stain density. (Edge detection circuits do not work well for cultures of HMSC because of the irregular densities and fuzzy edges of the colonies). Colony- forming efficiency was determined by direct count of colonies, usually with an inoculum of 100 cells per dish to minimize overlap of colonies.

Evaluation of differentiation. Qualitative visual esti- mates of myotube formation were obtained from the Giemsa stained plates that were included in all routine clonal growth assays. Quantitative data on extent of differentiation were obtained by spectrophotometric determination of creatine kinase specific activity in dense cultures. For the creatine kinase assays, a cell suspension was prepared with trypsin-EDTA as described above, counted, and diluted to 12,500-24,000 cells per ml in DS or SF media as indicated. Replicate 35 mm tissue culture petri dishes were inoculated with 2.0 ml aliquots of the cell suspension and the cultures were then grown to heavy confluency (typically 11 days without medium change).

The medium was then changed either to DME + 10pg/ml insulin (DMEI) to verify that the cultures had retained the ability to differentiate (40) or else to a medium that was being tested for its effects on differentiation. From 3 to 7 days after the medium change, the unfixed cells were rinsed twice with solution A, overlaid with 0.25 ml of 0.05M glycylglycine buffer at pH 6.75, frozen at -700 C, thawed, scraped from the culture surface, and vortexed, still in the glycylgly- cine buffer. Commercial kits were used to determine total creatine kinase activity (Sigma) and total protein (Biorad, Richmond, CA), and creatine kinase specific activity was calculated from the two values. In addition, creatine kinase isozyme distributions of selected cell lysates were examined with a commercial kit (Titan Gel-PC CPK- isozyme kit, Helena Laboratories, Beaumont, TX) to verify the relationship between increased specific activity and the presence of MB and MM isozymes.

RESULTS AND DISCUSSION

Sequence of studies. The results that are described below have been achieved by a combination of optimizing

the nutrient medium and replacing undefined supple- ments with more defined ones. These two types of experimentation were done concurrently. Most of the nutrient growth-response titrations leading to medium MCDB 120 were done with 5.0% dFBS and 0.5% dBPE supplementation during the same time period as replacement of dBPE and dFBS with more highly defined supplements was being studied in nutrient medium MCDB 131M. Thus, MCDB 120 and the serum-free supplements were not used together until near the end of the studies reported here.

Reduced serum and pituitary extract. The starting point for the current studies was growth medium GM-1 (7), which contained 15% fetal bovine serum and 0.5% chicken embryo extract in nutrient medium F10 (20). We found that bovine pituitary extract (BPE), which is an effective supplement for a variety of other cell types (9,24,25,43), at a concentration of 0.5% (70 ~g/ml protein) supported growth equivalent to that obtained with 0.5% embryo extract, and that dialyzed BPE (dBPE) was equally effective (data not shown). We also found that 5% dFBS plus 0.5% dBPE supported an adequate level of cfonal growth for nutrient growth-response studies, which are most effective when done with rate-limiting levels of dialyzed supplements (22).

Nutrient media. Although nutrient medium F10 supported good growth of HMSC with 5% dFBS and 0.5% dBPE, we tested a number of alternative media, including several from the MCDB series that had been optimized for clonal growth of other types of normal cells (23), to see if any would be better than F10 with these supplements. Among the media tested, MCDB 131 (29) was the best for clonal growth of HMSC. Medium F10 was generally a close second, and sometimes appeared to be equivalent to MCDB 131. However, we chose MCDB 131 as the starting point for our studies because it had a number of "modern" features that F10 lacked, such as a reasonably complete set of added trace elements. Other nutrient media tested and found not to be as effective as F10 and MCDB 131 for clonal growth of HMSC with 5% dFBS and 0.5% dBPE were MEM, DME, F12, DME:F12 (1:1), M199, McCoy's 5a, RPMI 1640, MB752/1, MCDB 110, MCDB 153, MCDB 170, and MCDB 202. Most studies in other laboratories on growth of skeletal muscle cells from various species, including humans, have been done with these nutrient media or with closely related formulations.

Magnesium. Medium MCDB 131 was developed specifi- cally for human microvascular endothelial cells derived from omental fat, and contains 10.0 mM Mg2?, which is optimal for those cells (29), but excessive for most other cell types. The clonal growth response of HMSC to varying amounts of magnesium (Table 3) indicated that the con- centration should be reduced. A modification of MCDB 131 with its magnesium ion reduced to 1.0 mM (MCDB 131M) was therefore selected as the nutrient medium to be used in detailed studies of qualitative and quantitative nutrient requirements for growth of HMSC.

Development of MCDB 120. The nutrients in MCDB 131M were omitted one at a time and added back over a wide range of concentrations to determine the optimal

HUMAN MUSCLE CELL SERUM-FREE MEDIUM 837

TABLE 3

RESPONSE OF HMSC TO MAGNESIUM ION'

MgSO 4 74H20 Total colony area lM/L) Imm2)

0 231 1.OE-5 228 1.0E-4 266 3.0E-4 462 6.0E-4 512 1.0E-3 (MCDB 131M) 525 2.0E-3 528 5.OE-3 529 1.0E-2 (MCDB 131) 437,

"Medium MCDB 131 was prepared without magnesium sulfate and supplemented with 5.0% dFBS and 0.5% dBPE. Magnesium sulfate was added back at the indicated concentrations. The inoculum was 1000 cells per plate. After incubation for 14 days, total area per dish covered by crystal violet stained colonies was determined as described in Materials and Methods.

amount of each for clonal growth of HMSC with 5% dFBS and 0.5% dBPE. Most components were found to be within the optimal range. However, the data indicated that arginine, methionine, threonine, pantothenate, and inositol should be increased to the levels in MCDB 120 (Table 1). In addition, although we could not see well defined requirements for iron, zinc, or copper in media containing both dFBS and dBPE, their levels in MCDB 131M were low enough to be potentially rate-limiting with defined supplements. We therefore elected to increase them to levels known to be effective for other cell types, and shown by our titrations not to be inhibitory for HMSC.

Subsequent testing in the serum-free medium de- scribed below has demonstrated clearly that iron was required (data not shown). The higher levels of copper and zinc in MCDB 120 also appeared to be marginally beneficial for serum-free growth, although a major requirement could not be demonstrated, probably due to

high background levels in other medium components. Individual growth-response titrations in MCDB 131M

also suggested that reductions in the concentrations of cysteine, glutamine, tryrosine, lipoic acid, and phenol red might be beneficial. However, when all of these reductions except phenol red were combined in a single medium, growth was reduced, both in MCDB 131M and in MCDB 120, possibly due to alternative requirements for higher levels of one or the other of the components. Since the effects were relatively small and appeared to involve complex balance relationships, we elected to proceed with studies on serum-free growth and differentiation without devoting more time to minor improvements in the nutrient medium. However, we did reduce the concentra- tion of phenol red to 3.3 X 10-6 M, which is consistent with most of the other MCDB media except MCDB 131 and MCDB 402.

In summary, MCDB 120 differs from MCDB 131 in its levels of arginine, methionine, threonine, pantothenate, inositol, iron, zinc, and copper, all of which have been increased, and in its level of phenol red, which has been

reduced. Medium MCDB 120 has consistently supported better clonal growth of HMSC than MCDB 131M, both with serum-free supplementation and with low levels of serum and embryo extract. However, in all cases that have been examined, qualitative requirements for defined and/or undefined supplements remain the same in both media.

Replacement of dBPE. Pituitary extract is a known source of fibroblast growth factor (FGF), and FGF is known to stimulate growth of mononucleate muscle cells from various mammals and to inhibit their differentiation (18,19,32,33,34,36). Commercial preparations of basic FGF (30 ng/ml) fully replaced the requirement of HMSC for dBPE in a background medium of MCDB 131M (or MCDB 120) plus 5% dBFS (Figure 1). In preliminary tests, high purity full-length acidic FGF (prostatropin, ref. 10) supported optimal growth at 3 ng/ml (data not shown). We have not yet tested basic FGF of comparable purity, but literature reports suggest that it will be active at even lower concentrations (18,38).

Askanas and Gallez-Hawkins (3) have reported that addition of a combination of FGF, EGF, and insulin to the culture medium for HMSC eliminates the requirement for embryo extract and reduces the amount of serum needed for growth, differentiation, and partial matura- tion. Although they did not specifically identify which component replaced the need for embryo extract, their results appear to be in agreement with ours, both with

regard to FGF, and with regard to EGF, which is discussed below.

Serum-free medium. In the presence of commercial basic FGF, we were able to replace dFBS with the following mixture of defined and semi-defined supple- ments: bovine insulin, 3.OE-5 M (180 j.g/ml); mouse EGF, 10 ng/ml; dexamethasone, 1.OE-6 M; BSA, 0.5 mg/ml, and bovine fetuin (Pederson), 0.5 mg/ml. These five serum-replacing components (Table 2) have been desig- nated "SF". When medium MCDB 131M is supplemented with SF plus FGF, the resulting serum-free growth is at least as good as that obtained with 5% dFBS and 0.5%

800 E - bFGF (Commercial) E

600 "

>, 400 ':

dBPE

_o / 200

0 '' 0 I 10 102 03 I04 10

ng /ml

FIG. 1. Replacement of dBPE with basic FGF. Medium MCDB 131M was supplemented with 5% dFBS plus the indicated amounts of dBPE or basic FGF (BRL 6111-LA). Each 60 mm petri dish was inoculated with 200 HMSC and incubated for 14 days without medium change. The colonies were then fixed and stained and total colony area per dish was determined as described in Materials and Methods.

838 HAM ET AL.

400

E 300 E

, 200 0

* 100 o-

Ike 4 1 ' FIG. 2. Effect of deletion of individual components of SF +

FGF. Clonal cultures of HMSC were inoculated into MCDB 131M supplemented with complete SF (Table 2) plus 30 ng/ml FGF, or the same mixture with individual components omitted. Total colony area per dish was determined after 14 days of growth, as described in Materials and Methods.

dBPE. Growth in MCDB 120 is slightly better in all cases, but again growth with SF plus FGF is equivalent to that with 5% dFBS plus 0.5% dBPE.

As shown in Figure 2, removal of any one of the five

components of SF from a mixture of SF + FGF reduces

growth. There is sometimes also a reduction when FGF is removed, but the requirement for FGF in the presence of SF is far less stringent than with dFBS as the only other

supplement. Because of the high cost of FGF and the minimal benefit from its presence, FGF was not included

in the final formulation of serum-free growth supplement SF, and many of the experiments described below have been done without FGF. These data stand in sharp contrast to the major role that FGF appears to play in

promoting growth and suppressing differentiation in

myogenic mouse cell lines (32-35). The five components of SF have all been employed in

other serum-free or reduced serum media for muscle cells or muscle cell lines from various species (1,4,11,14,16,18,31,45). In particular, insulin, dexametha- sone and fetuin were included in medium MM-1 by Florini and Roberts (16) and have been frequent components of more recent formulations.

Dexamethasone. Dexamethasone is presumed to act by stimulating glucocorticoid receptors. We have been unable to obtain equivalent growth with hydrocortisone, even at higher concentrations, but further study is needed before

drawing firm conclusions from these preliminary observations (data not shown).

Fetuin. The fetuin used in our serum-free medium is

prepared commercially (Sigma Type III) by the proce- dure of Pederson (39). It is a relatively crude ammonium sulfate fraction of fetal bovine serum protein, and cannot be considered to be fully "defined" in the strictest sense of the term. Hence, we generally refer to media

containing it as "semi-defined" or "serum-free". In

preliminary testing, fetuin prepared commercially (GIB- CO) by the more rigorous procedure of Spiro (42), was not active in our test system. However, we have not yet tested

Spiro-type fetuin that has been subjected to exhaustive

dialysis against chelating agents, which has been

reported (16) to remove inhibitory heavy metal residues that are introduced during the purification process.

TABLE 4

RESPONSE TO SERUM ALBUMIN'

Colony area Species Description Source (mm 2n

Control, No Albumin 97 Bovine Fraction V Sigma A-4503 557 Bovine Fraction V Sigma A-7888 497

RIA grade Bovine Fraction V Sigma A-6003 612

Fatty Acid Free Bovine Crystalized Sigma A-4378 702 Bovine Fatty Acid Free Sigma A-7030 701

RIA grade Bovine Globulin-free Sigma A-0281 442

Fatty acid free Human Crystalized Sigma A-9511 830 Human Fraction V Sigma A-1887 1,047

Fatty Acid Free Human Crystalized, Fatty Sigma A-3782 821

Acid and Globulin Free Pig Fraction V Sigma A-2764 1,109 Horse Fraction V Sigma A-9888 1,502 Sheep Fraction V Sigma A-3264 594 Bovine 5% dFBS 1,829

aClonal cultures were inoculated with 500 cells per 60 mm dish in MCDB 131M SF prepared without BSA and supplemented with the indicated albumin at 0.5 mg/ml. Total colony area per dish was determined as described in Materials and Methods after a 14 day growth period without medium change.

HUMAN MUSCLE CELL SERUM-FREE MEDIUM 839

We have begun a detailed study of the roles played by Pederson-type fetuin in promoting clonal growth of HMSC in a culture medium that is otherwise quite highly defined. At the present time, we have data suggesting that at least three separate activities may be involved, including neutralization of residual trypsin and supply- ing polyamines (21). However, these findings are still too

preliminary to present in detail. Serum albumin. In the defined medium for rat muscle

satellite cells developed by Allen et al. (1), BSA is described as a carrier for linoleic acid. However, in our culture system, BSA is highly stimulatory to clonal growth of HMSC without added linoleic acid and

cohmmercially delipidated BSA preparations are equally active (Table 4). We have not yet undertaken a detailed

study of lipid metabolism or lipid requirements of HMSC. However, our current serum-free medium with

delipidated BSA supports extensive clonal growth of HMSC with no obvious sources of preformed fatty acids, other than possible contaminants in fetuin or other medium components.

BSA preparations that are certified for use in insulin RIA assays and crystalline BSA preparations that are described as "essentially globulin-free and essentially fatty acid free" have almost the same activity as ordinary

BSA. This suggests that the albumin itself may be

required. However, BSA is known to bind many different

ligands tightly, and supposed requirements for serum albumin have in the past usually proven to be

requirements for other substances carried on the serum albumin. Thus, further study is needed to determine the exact nature of the albumin requirement exhibited by HMSC in the current serum-free medium.

In recent experiments, we have observed that human, pig, and horse serum albumin preparations often support substantially better serum-free growth than bovine serum albumin (Table 4). We have continued to employ RIA grade BSA because all of our preliminary experiments were done with it and because we still have unfinished

problems to resolve concerning the insulin requirement (see below). However, current data strongly suggest that clonal growth of HMSC in the serum-free medium could be improved further by use of alternative serum albumin preparations. The intense growth stimulation that resulted from use of 5% dFBS as a source of BSA (Table 5) is discussed below under "doubly-supplemented media".

Insulin. Insulin is thought to stimulate rat muscle cell growth largely by cross reaction with receptors for the insulin-like growth factors (12,13,15,17). Because of this,

TABLE 5

CREATINE KINASE SPECIFIC ACITIVITYa

Creatine Kinase (mU/mg)

No collagen With collagen

Growth medium Test medium 3 days 7days 7 days

Experiment A: MCDB 131M DS DME + I 574 1,104 1,569 MCDB 131M DS DME SF 272 271 -

MCDB 131M DS DME DS 369 424 367 MCDB 131M DS DME + 2% HS 500 818 903 MCDB 131M DS MCDB 131M + I 572 870 975 MCDB 131M DS MCDB 131M DS 218 236 536 MCDB 131M DS MCDB 131M SF 348 352 602

Creatine Kinase (mil/mg) With collagen

Growth medium Test medium 6 days

Experiment B: MCDB 131M DS DME + I 1,830 MCDB 131M DS DME alone 1,295 MCDB 131M DS F12 + I 1,055 MCDB 131M DS F10 + I 1,020 MCDB 131M DS MCDB 131M + I 1,700 MCDB 131M DS MCDB 131M DS 440

Creatine Kinase (mU/mg)

No Collagen With collagen

Growth medium Test medium 6 days 6 days

Experiment C MCDB 120 SF DME + I 556 801 MCDB 120 SF MCDB 120 SF 142 216 MCDB 120 DS DME + I 1,021 1,205 MCDB 120 DS MCDB 120 DS 221 324

"Cultures were inoculated with 25,000 cells per 35 mm dish in the indicated growth media and grown for 11 days (MCDB 131M DS and MCDB 120 DS) or 14 days (MCDB 120 SF) with medium changes every 3-4 days. The growth medium was then replaced with the test medium. After 3-7 days, as indicated, creatine kinase specific activity was determined as described in Materials and Methods. Tissue culture plastic petri dishes were used either uncoated or coated with collagen, as indicated.

840 HAM ET AL.

the levels of insulin in culture media for muscle cells are

frequently at non-physiological levels. The amount of insulin that we have found to be optimal for serum-free

growth of HMSC is unusually high, even in comparison to other muscle cell media. In addition, the requirement tends to be erratic, with substantial growth sometimes occurring in the absence of added insulin. Thus, further studies on the role of insulin and possible replacement of insulin with other substances are clearly needed.

Epidermal growth factor. EGF is mitogenic to a wide range of cell types, but has generally not been found to be stimulatory to non-human muscle cells. Dollenmeier et al. (14) reported that EGF promoted growth of contamin-

ating fibroblasts, but had little effect on embryonic chicken myoblasts. Allen et al. (1) found that EGF did not promote growth or differentiation of rat satellite cell cultures. Lim and Hauschka (32,34) found that EGF is not mitogenic to the mouse MM14 cell line, although these cells possess EGF

receptors during the growth phase and lose them during commitment to the terminal differentiation, which is triggered by withdrawal of FGF. However, there is a differentiation- deficient variant of MM-14 designated DD-1, which does not require FGF for growth with 15% serum. Under low serum conditions, growth of DD-1 cells can be stimulated either with EGF or with FGF (32,34). Differentiation-deficient mouse muscle line BC3H1 also exhibits a limited mitogenic response to EGF (18).

The effects of EGF on human muscle cells may be different than for muscle cells from other species that have been studied. Askanas and co-workers (3,5) have shown that EGF acts synergistically with FGF and insulin to promote growth, differentiation, and some aspects of maturation of cultured normal human muscle cells. EGF

has been included in a serum-free medium for human muscle cells described in an abstract by Askanas et al (4), but not yet published in detail. Lim and Hauschka (32) also mention the EGF binding capacity of early passage human myoblasts, but do not elaborate on its possible role. These reports, together with our data showing that EGF is needed for clonal growth of HMSC in our serum-free medium, suggest that there could be a species-based difference between HMSC and myoblasts or satellite cells from other vertebrate species with regard to the requirement for EGF. Species differences in

receptor specificity are probably not involved, since mouse EGF is fully active for human muscle cells but does not stimulate the mouse MM-1 cell line. However, before firm conclusions are drawn, cells from other species need to be tested under conditions comparable to those we are using for HMSC.

Optimization for growth. The current semi-defined serum-free medium for normal HMSC represents the first time that a dual approach involving both systematic optimization of the nutrient medium (22) and replacement of undefined supplements with hormones, growth factors, and other more defined substances (6) has been

applied to growth of skeletal muscle cells from any species. Previous defined media for normal HMSC (3,11,45) have utilized either DME or F14 (44) as the nutrient medium without further optimization. These defined media have also favored differentiation of HMSC rather than optimal growth. In contrast, our serum-free medium strongly favors growth, with relatively little fusion and myotube differentiation. However, as will be described below, cells grown in our serum-free medium retain the capacity to fuse and differentiate when

FIG. 3. Growth stimulation in doubly supplemented medium. Petri dishes (60 mm) were inoculated with 500 HMSC each, incubated for 14 days, fixed with methanol, and stained with crystal violet as described in Materials and Methods. A. Colonies formed in MCDB 120 supplemented with 5% dFBS and 0.5% dBPE; B. Colonies formed in MCDB 120 supplemented with SF + FGF; C. Colonies formed in MCDB 120 supplemented with SF + FGF + 5% dFBS.

HUMAN MUSCLE CELL SERUM-FREE MEDIUM 841

transferred to an appropriate differentiation-promoting medium.

Direct plating into serum-free medium. Another major advantage of our current serum-free medium is that we can routinely inoculate freshly trypsinized HMSC at clonal density directly into it. In previously described studies from other laboratories, the cells have generally been seeded into serum-containing media and then transferred to serum-free conditions after the cells have attached and become stabilized.

Doubly supplemented media. Clonal growth of HMSC in MCDB 120 or MCDB 131M supplemented with SF is consistently as good as with 5% dFBS plus 0.5% dBPE in the same nutrient medium. However, when 5% dFBS is added on top of SF with or without FGF, clonal growth of normal HMSC in the resulting doubly supplemented (DS) media is greatly enhanced (Figure 3). Colony forming

2000-

L I FIO MCDB 120

1500

E

S1000

500

-6

100 -

o\?\ kk \O\*

6

' 9

' \* \ \*

FIG. 4. Comparison of clonal growth of HMSC in medium F10 and medium MCDB 120 with various supplements. Each 60 mm petri dish received an inoculum of 200 HMSC added to 5.0 ml of F10 or MCDB 120 supplemented as indicated. After 14 days without medium change, the colonies were fixed and stained and total colony area per dish was determined as described in Materials and Methods.

efficiency is approximately 30% and the colonies that are formed are uniformly large and healthy appearing. Clonal growth in MCDB 120 DS (or MCDB 131M DS) substantially exceeds that previously obtained in conven- tional media with pituitary (or embryo) extract and much higher levels of serum, and only a modest further

improvement can be obtained by increasing the level of serum in the DS medium to 15% (Figure 4). Growth of monolayer cultures is also excellent in DS media, and we now routinely grow our stock cultures of HMSC in MCDB 120 DS (with 5% dFBS).

Primary cultures. We have been able to establish primary clonal cultures directly in MCDB 131M DS or MCDB 120 DS without the need for conditioning of the medium or collagen coating of the wells, which are important features of the procedure we were using previously (7). Initial growth rates are slightly faster in conditioned GM-2 than in MCDB 131M DS or MCDB 120 DS without conditioning. However colony-forming effic- iencies are equivalent, and all of these media yield large numbers of clonal cultures that can readily be grown up to confluent monolayers and stored frozen.

Differentiation. Because these studies were done with cloned human muscle satellite cells, we have been able to focus exclusively on improvement of growth, with no immediate concern about expression of differentiated properties or overgrowth by fibroblasts. The net result has been the development of media that strongly favor growth of HMSC at the expense of differentiation. When growth in MCDB 120 or MCDB 131M with either SF or DS supplementation, the cells in our cultures tend to be highly elongated and quite fibroblastic in appearance. Careful examination of large colonies with Giemsa staining generally reveals a few multinucleate myotubes, and some additional fusion occurs when cultures are plated at higher densities and allowed to remain confluent with periodic refeeding.

The low level of differentiation in the new growth media makes it necessary to ask whether the cells may have lost their ability to fuse and express muscle-specific proteins. Evidence that this is not the case has been obtained by growing cultures to confluency in the growth media and then transferring them to a medium that favors differentiation, such as DME supplemented with 10 jg/ml insulin (DMEI), which was used by Pinset and Whalen (40) for similar studies on rat L6 cells.

Within 3 days after transfer to DMEI, the cells undergo extensive fusion to form multinucleate myotubes (Fig. 4). This is accompanied by a major increase in creatine kinase specific activity (Table 5). Preliminary electro- phoretic studies indicate that most of the increase is due to enhanced levels of the MM and MB isozymes of creatine kinase (data not shown). Spontaneous twitching has also been observed in cultures allowed to remain in DMEI for six days after transfer from DS growth media. Control cultures that are transferred into fresh DS or SF media show very little fusion or increase in creatine kinase specific activity.

We are also able to examine the ability of media to support differentiation by replacing the growth medium

842 HAM ET AL.

FIG. 5. Comparison of growth and differentiation media. Inocula of 48,000 cells per 35 mm dish were grown for 11 days in MCDB 120 SF, generating heavily confluent cultures of elongated fibroblastic appearing cells. The medium was then changed to fresh MCDB 120 SF (left) or to DMEI (right) and the cultures were incubated for another 6 days. Phase contrast pictures of the living cells were then taken with a Nikon Diaphot-TMD inverted phase contrast microscope (103X).

in confluent cultures with appropriate test media. Assays of this type have shown that insulin in excess of that carried over from the growth medium is not strictly needed for differentiation of HMSC, although the level of creatine kinase obtained in unsupplemented DME remains somewhat less than in DME plus insulin (Table 5). The extent of differentiation in DME plus 2% horse serum, which has been used as a differentiation- promoting medium by many previous investigators, is also less than that in DMEI.

MCDB 131M and MCDB 120 are only slightly less supportive of differentiation than DME. Cultures trans- ferred into these media plus insulin exhibit substantial fusion and creatine kinase synthesis, but not as extensive as in DMEI. Intermediate levels of differentiation are also observed in F10 or F12 plus insulin. Collagen coating of the dishes somewhat increases the level of creatine kinase synthesis in most media, but is not essential for differentiation of HMSC.

Differentiation is inhibited by SF both alone and in combination with 5% dFBS, both in DME and in the MCDB media. Further studies will be needed to identify the specific combinations of components that are responsible for the inhibition. Our initial experiments suggest that complex interactions may be involved. In addition, FGF, which is not present in serum-free supplement SF, may be of lesser importance in control of differentiation of HMSC than in the mouse MM-14 myoblast cell line (35).

Conclusions. We strongly recommend a sequential two medium approach to growth and differentiation of HMSC. Our studies, which were done with clonal cultures and allowed optimization of the medium for growth without concern about differentiation or overgrowth by other cell types, have verified that conditions that are optimal for growth of HMSC do not favor their differentiation. In addition, the limited studies that we have completed on conditions that favor differentiation make it appear unlikely that culture conditions will be

found that promote high levels of both growth and differentiation, which is to be expected, since extensive fusion removes large numbers of mononucleate cells from the proliferating pool.

Many questions remain unanswered about the growth requirements of HMSC, and extensive further study is needed to develop more completely defined media and to improve growth and differentiation in those media. However, the media described in this paper already provide many advantages over existing serum-free and low-serum culture media for HMSC. The availability of optimized nutrient media that have been developed specifically for growth of HMSC will greatly facilitate use of these cells as model systems for the study of diverse aspects of normal and abnormal muscle development and function. In addition, the ability to grow HMSC in a semi-defined medium, followed by differentiation in a highly defined medium, will open the way for many lines of research that could not readily be pursued in media containing large amounts of whole serum and other undefined additives.

NOTE ADDED IN PROOF

Figure 1 shows that FGF supported clonal growth of HMSC with 5% dialyzed serum and Figure 2 shows that FGF failed to replace EGF in the serum-free supplement, SF. A more detailed study has shown that instability of FGF in serum-free media, but not in media with serum, caused at least part of this apparent discrepancy. When stabilized with 1.0 yg/ml heparin, commercial basic FGF at 30 ng/ml supports sub- stantial serum-free growth of HMSC in the absence of EGF, although growth with EGF, or with EGF plus stabilized FGF, is usually better.

Also, a report has been called to our attention showing that rat muscle cell lines exhibit a biphasic response to insulin and IGF-I, with stimulation of growth continuing at high levels that inhibit differentiation, although lower levels are

specifically needed for differentiation (Florini, J. R.; Ewton,

HUMAN MUSCLE CELL SERUM-FREE MEDIUM 843

D. Z.; Falen, S. L.; Van Wyk, J. J. Biphasic concentration

dependency of stimulation of myoblast differentiation by somatomedins. Am. J. Physiol. 250:C771-C778; 1986).

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This research was supported by a grant from the Muscular Dystrophy Association. We thank Lisa Pieti for excellent technical assistance.

EDITOR'S STATEMENT

This article describes the optimization of both the basal nutrient medium and growth factor

requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne Muscular

Dystrophy.