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Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture Angie Rizzino, Ph. D., Heather Rizzino, B.Sc. and Gordon Safo, Ph.D. ecent studies have suggested that the nutri- R tional and hormonal requirements of mam- malian cells in culture are interdependent. Thus far such relationships have been examined only superficially. One of the aims of this review is to focus attention on the feasibility of exam- ining this and other related problems. In this article we provide an overview of the nutritional and hormonal requirements of ani- mal cells in culture. To a large extent this in- formation has been derived from studies con- cerned with the formulation of defined media. To support this point, and to situate the reader, we first give a summarized history of cell cul- ture as it relates to defined media. In the fol- lowing two sections we discuss the nutritional and hormonal requirements of cells, giving particular emphasis to the most recent develop- ments. In the final section we discuss ap- proaches to several questions that are related to the nutritional and hormonal requirements of cells. conducting physiological studies. As early as 1911 it was obvious to Lewis and Lewis’ that media of known composition would be superior to complex undefined media for this purpose. At a time when tissue culture was conducted in plasma clots, Lewis and Lewis studied tis- sue explants in simple salt solutions. They found that cell survival was improved by the addition of glucose and amino acids (unspeci- fied). Unfortunately for those of us interested in the requirements of cells, the importance of their findings was overshadowed by the work of Carrel. In 1913 Carrel2 discovered that the addition of embryo extract to plasma dramatically in- creased the growth of tissue explants. For several decades most investigators em- ployed modifications of Carrel’s culture me- dium and no attempts were made to develop defined media. Although there were sporadic attempts to replace embryo extract with known components, which included hormones, amino acids and vitamins, the need for these components was not systematically studied.3-5 Early History of Defined Media In the early 1900’s Several investigators began to study tissue explan& with the idea of In 1946, White6 developed the first defined medium. It supported the prolonged Survival but not active growth of tissue explants. White of the nutritional requirements of animals, plants and microorganisms, His medium con- tained glucose, salts, amino acids and vita- contained the seven vitamins and 11 d the 13 Dr. Rizzino iS Research BiO/OgiSf and Dr. Sat0 IS based his medium on the current knowledge Professor of Biology at the University of California, San Diego, La Jolla, CA 92093. Ms. Rizzino is a technical and editorial assistant to Dr. Rizzino. A limited number of reprints of this article may be obtained mins. It is interesting to note that his medium from Dr. Rizzino. There are no reprintsof unsigned reviews. NUTRITION REVIEWSIVOL. 37. NO. 12iDECEMBER 1979 369

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Page 1: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

Defined Media and the Determination of Nutritional and Hormonal Requirements of

Mammalian Cells in Culture

Angie Rizzino, Ph. D., Heather Rizzino, B.Sc. and Gordon Safo, Ph.D.

ecent studies have suggested that the nutri- R tional and hormonal requirements of mam- malian cells in culture are interdependent. Thus far such relationships have been examined only superficially. One of the aims of this review is to focus attention on the feasibility of exam- ining this and other related problems.

In this article we provide an overview of the nutritional and hormonal requirements of ani- mal cells in culture. To a large extent this in- formation has been derived from studies con- cerned with the formulation of defined media. To support this point, and to situate the reader, we first give a summarized history of cell cul- ture as it relates to defined media. In the fol- lowing two sections we discuss the nutritional and hormonal requirements of cells, giving particular emphasis to the most recent develop- ments. In the final section we discuss ap- proaches to several questions that are related to the nutritional and hormonal requirements of cells.

conducting physiological studies. As early as 1911 it was obvious to Lewis and Lewis’ that media of known composition would be superior to complex undefined media for this purpose. At a time when tissue culture was conducted in plasma clots, Lewis and Lewis studied tis- sue explants in simple salt solutions. They found that cell survival was improved by the addition of glucose and amino acids (unspeci- fied). Unfortunately for those of us interested in the requirements of cells, the importance of their findings was overshadowed by the work of Carrel.

In 1913 Carrel2 discovered that the addition of embryo extract to plasma dramatically in- creased the growth of tissue explants. For several decades most investigators em- ployed modifications of Carrel’s culture me- dium and no attempts were made to develop defined media. Although there were sporadic attempts to replace embryo extract with known components, which included hormones, amino acids and vitamins, the need for these components was not systematically studied.3-5 Early History of Defined Media

In the early 1900’s Several investigators began to study tissue explan& with the idea of

In 1946, White6 developed the first defined medium. It supported the prolonged Survival but not active growth of tissue explants. White

of the nutritional requirements of animals, plants and microorganisms, His medium con- tained glucose, salts, amino acids and vita-

contained the seven vitamins and 1 1 d the 13

Dr. Rizzino iS Research BiO/OgiSf and Dr. Sat0 IS based his medium on the current knowledge Professor of Biology at the University of California, San Diego, La Jolla, CA 92093. Ms. Rizzino is a technical and editorial assistant to Dr. Rizzino.

A limited number of reprints of this article may be obtained mins. It is interesting to note that his medium from Dr. Rizzino. There are no reprintsof unsigned reviews.

NUTRITION REVIEWSIVOL. 37. NO. 12iDECEMBER 1979 369

Page 2: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

amino acids shown by Eagle in 1955 (see below) to be essential for the growth of cultured cells.7 Although lacking certain essential nutri- ents, White’s medium demonstrated the feasibility of developing defined media and of identifying the nutritional requirements of cells. Shortly afterwards (1950) Morgan et a1.8 de- veloped a similar but more complex defined medium, medium 199.

Concurrent with these studies was the devel- opment of the first permanent cell lines as we know them today.93’0 For the first time it was possible to work with a uniform and reproduc- ible population of cells. This altered the course of cell culture dramatically and the emphasis rapidly switched from tissue explants to bona fide cell cultures. By using the newly estab- lished cell line (L cells) and modifying the media of White and Morgan et al., several investi- gators were able to develop the first defined media that would support active cell growth.’ 1-13

For the reader interested in the early history of cell culture (1907 to 1950) we suggest three reviews.14-16 The period of 1946 to 1972 gets excellent coverage by Higuchi.”

The Nutritional Requirements of Animal Cells in Culture

Much of our current knowledge of the nutri- tional requirements of cells stems from the work of Eagle. In 1955 Eagle published several classic papers dealing with the amino acid and vitamin requirements of cells in the presence of dialyzed serum. He demonstrated that L and HeLa cells require 13 amino acids (argi- nine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine) and seven vitamins (biotin, folic acid, nicotinamide, pantothenic acid, pyridoxal, thiamine and ribo- flavin, or equivalent forms).7 These findings formed the basis of Eagle’s now famous mini- mum essential medium (MEM).lB From a nu- tritional point of view, both the main strength and the main weakness of Eagle’s work was his use of dialyzed serum. Although dialyzed serum enabled Eagle to detect specific require- ments for certain amino acids and vitamins, it also masked the need for other essential nu- trients, which were present in the dialyzed serum (for example iron specifically bound to

370 NUTRlTlON REVIEWSIVOL. 37, NO. 12iDECEMSER 1979

transferrin). It is obvious that problems of this nature can ultimately be avoided if serum is replaced with highly purified serum factors.

In the mid-1950’s several defined media were developed for the growth of L cell^.^^-^^ The media contained a variety of components that together enhanced cell growth. These stud- ies were concerned with obtaining good growth rates but not with identifying the spe- cific requirements of cells. Over the next 20 years the practice of using complex “rich” media became the rule rather than the excep- tion. During that period many complex defined media were developed. Due to their complex- ity a large proportion of these media were able to support the growth of many different cell types, regardless of tissue or species origin. Consequently, the nutritional requirements of specific cell types were scarcely examined.

Despite this situation, since the late 1950’s the use of defined media has been largely re- sponsible for the progress made in identifying additional nutritional requirements. A good example is the work of Ham and coworkers. In the course of developing the well-known medium F-12, they identified the need for fatty acids (linoleic acid), polyamines (putrescine) and trace elements ( s e l e n i ~ m ) . ~ ~ - ~ ~

As a result of Eagle’s work and studies with defined media, many nutrients are now known to be essential for one or more cell types. We have summarized this information in Table I and have commented on these requirements. In particular we have pointed out whether a nutrient is required by all, most or some cells. It should be noted that fatty acids and trace elements were the main constituents missing from the early work on the nutritional require- ments of cells. We expect the number of essential nutrients to increase as work with nor- mal cell lines and primary cultures increases and as purer components for culture media become available.

Since the major essential nutrients for cells in culture have been identified, it is obvious that attention must now be focused on the re- quirements of individual cell types. Recently Ham, McKeehan and coworkers have at- tempted to tackle this problem by systemati- cally developing defined media that are opti- mized for a particular cell type.*5~36-~* Thus

Page 3: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

TABLE 1. Nutrients Known to Be Required by Cells in Culture"

V

Nutrient

Sugars Glucose

V

Pyruvate

Amino acids lsoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Arginine Cyst(e)ine Glutamine Histidine Tyrosine

Serine Alanine Aspartic acid Glycine Glutamic acid Proline

Vitamins Biotin Folic acid

Niacinamide Pantothenic acid Pyridoxine

Ri bof lavi n Thiamin 8 1 2

Ascorbic acid Vitamins A,D & K

Vitamin E

Retinoic acid

Fatty acids Linoleic and

Oleic acids Other organics Cholesterol

Special Comments References

Can be replaced by fructose, mannose, maltose or galactose for some cells.

Z 2

Required by some mouse embryonic cells. 17.22.25

Early development of mouse embryos, up to and including trophoblast outgrowth, requires all these amino acids with the exception of isoleu- c1ne.2~

7.22.25

Some are required by particular cell types. In addition all appear to be necessary for growth at low density.85

17.22.25.26

17.22.25

Can be replaced by a combination of glycine. a purine & thymidine

7.17.22.25

In one case can be replaced by six non-essential amino acids.

7 z 2 5

Shown to affect fatty acid metabolism 28 17 27

17 25 29

Appear not to be essential for the cells that have been carefully studied in serum-free media Does not affect cell growth but is required for certain cell functions 30

z5

Affects both growth and differentiation 31 52

Sometimes required in combination with fatty acid-free serum alb~min.3~.~3

l7

Required by primary diploid human fibroblasts and a cell line of porcine kidney origin.17

NUTRIT ION REVIEWSIVOL. 37. NO. 12IDECEMBER 1979 371

Page 4: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

TABLE I: Continued -

Nutrient Requirement Special Comments References

Choline lnositol Pu trescine

Bulk ions Sodium Potassium Calcium Magnesium Chloride Phosphate Bicarbonate

(or C02)

Trace elements Iron

a nc Selenium Copper Manganese Molybdenum Vanadium

This is true when the population is sparse or when loss of COP is not inhibited.25

Uptake of iron is usually mediated by trans- ferrin.3’

22.25

22.25

17.34

25.35

17

17.25

21.34

2 5

- this table lists most but not necessarily all of the nutrients currently considered to be required by cells

- this or equivalent molecules, for example, folinic acid can replace folic acid - a natural metaboliteof vitaminA

in culture

far they have developed different media for human, chicken, Chinese hamster, mouse and duck fibroblasts. Since their approach is a good method for studying the nutritional re- quirements of cells, we will summarize it here. The synthetic portion of the medium is modi- fied so that in the presence of dialyzed serum cell growth occurs. Once this is accomplished, the concentration of dialyzed serum is reduced to a level that will barely support growth. The concentration of each component is then indi- vidua!ly raised and lowered in order to deter- mine its optimum concentration. Once cell growth has been restored by optimizing the concentration of each component, the level of dialyzed serum is reduced further. The entire procedure is repeated several times until growth can no longer be restored by adjusting the concentrations of the components.

372 NUTRITION REVIEWSNOL. 37, NO. 1ZiDECEMBER 1979

Using this approach it should be possible to develop media optimally suited to the nutritional needs of any particular cell. For example, the medium MCDB 105 has been developed for a normal human cell line, WI-38. It contains 56 components, including 20 amino acids, nine vitamins and 11 trace elements.39 This me- dium, plus 1 percent dialyzed serum, is able to support the clonal growth of WI-38 cells. Unfortunately the published details of this me- dium do not permit one to readily distinguish between those components that are essential for growth and those that are present to mini- mize the “biosynthetic load” of the cell. Lastly, in order to be certain that all of the nutritional requirements have been identified it will be necessary to replace the residual serum with highly purified serum factors. In the next sec- tion we discuss the approach taken by mem-

Page 5: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

bers of our laboratory to identify these factors. Ultimately the combination of the two ap- proaches should permit the identification of all requirements of a given cell type.

The Hormonal Requirements of Animal Cells in Culture

Before beginning this section we would like to point out that we will use the term hormone to include not only classical hormones, such as insulin, but also the so-called growth fac- tors that have been discovered during the past 30 years. Until their mechanisms of action have been elucidated we fail to see the need to use two different terms to artificially classify these molecules.

As discussed in the previous section, the development of permanent cell lines and "rich" culture media enabled many laboratories to successfully develop defined culture media for a variety of established cell lines. Although these pioneering studies suggested that al- most any cell type could be grown in defined media, virtually all of the early work, and many of the present day studies, suffer from one or more of the following weaknesses: 1) a period of adaptation was required for growth in de- fined media, 2) transformed (tumorigenic) cells were used, and 3) the media were devoid of hormone supplements. In regards to the last point, only a few notable exceptions are found in the early l i t e r a t ~ r e . ~ ~ ? ~ ' As a direct conse- quence of these weaknesses, little attention was focused on the hormonal requirements of

Recognizing the need to identify the hor- monal requirements of cells, this laboratory took a different approach based on several assumptions. We assumed, and it is now generally accepted, that all normal cells and most transformed cells require serum factors in order to grow in culture. We also assumed that one of the primary roles of serum was to provide hormones - different ones for different cell types.44 We now know that this assump- tion is also true. By applying the above logic, this laboratory has succeeded, over the past four years, in developing hormone-supple- mented defined media for a wide variety of mouse, rat and human cell lines (Table 1 1 ) .

As in the study of nutritional requirements,

cell s ,174 2 4 3

the best approach for identifying hormonal requirements uses culture media whose serum concentration has been reduced and thus cannot support significant cell proliferation. Purified hormones (and other purified serum factors), either individually or in various com- binations, are then added to this maintenance medium. Those factors found to restore growth are then included in the medium and the concentration of serum is again reduced to a level unable to support growth. This pro- cess of hormone additions and serum reduc- tion is repeated until the serum is completely eliminated or no further improvements can be obtained by the addition of known factors. By the use of this approach, or minor modifications of it, this laboratory has developed hormone- supplemented defined culture media for more than ten different cell lines (Table 11).31.45.46

In those cases where serum cannot be totally eliminated, it should be possible to isolate and purify the few factors involved, using the cells in question to monitor the purification.

An examination of Table II indicates that each cell type requires a unique combination of serum factors. These fall into several classes: hormones such as insulin and epidermal growth factor (EGF), attachment factors such as fibronectin (Clg), transport proteins such as transferrin and, for some cell lines, nutrients such as putrescine and retinoic acid. Thus far all cells have been shown to require both insulin and transferrin, while several cell lines also re- quire fibronectin as an attachment factor. Al- though we cannot discuss the function of these molecules in detail, we will briefly comment on transferrin, fibronectin and insulin since they represent the major classes of required serum factors.

Transferrin is believed to function as an iron transport protein. It is known that the iron- transferrin complex must bind to the cell in order to supply the cell with iron. Its fate there- after, however, appears to differ with the cell type and this is still under investigation. The role of fibronectin is proving to be quite impor- tant. Fibronectin in vivo is believed to play a role in cell-cell interactions as well as in cell interactions with various extracellular ma- trices.57 Furthermore, the appearance of fibronectin is developmentally regulated and

NUTRITION REVIEWSNOL. 37, NO. 1 PIDECEMBER 1979 373

Page 6: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

Table II. The Serum Factor Requirements of Established Cell Lines Studied in Our Laboratory

Cell Origin Refer- I TF Clg EGF FGF Additional Requirements Line ences (pgiml) (pgiml) (pglml) (nglml) (ng/ml)

~ ~ ~ ~~~~ ~~ ~

GH3 rat pituitary carcinoma

GC

HeLa human cervical carcinoma

M2R mouse melanoma

MDCK dog kidney

Fg mouse terato- carcinoma

RF-1 rat ovary

6104 rat neuro- blastoma

MCF-7 human mammary carcinomd

TM4 Sertoli cells

T84 human colon carcinoma

47.48

45

4 9

50

51

52

53.54

34.55

56

31

29

5

5

5

5

5

1

2

5

0.1

5

2

5

5

5

0.5

5

5

5

100

25

5

2

5

8

10

7.5

1 TRH (1 nglml), T3 (100 nu) , PTH (0.5 ngiml), SM-C (1 ngiml)

As for GH3 except that SM-C is not needed.

1

10 50 HC (50 nM)

Testosterone (10 nM), FSH (0.4 pgiml), LHRH (10 ngiml). NGF (3 ng/ml)

HC (50 nM), T3 (.005 nM), PGEi (25 ngiml)

HC (10 nM). T3 (0.3 nM)

Progesterone (20 nM). putrescine (100 pM)

100 PGF2<, (100 ngiml), a-1 Spreading Protein (1 pgiml)

3 FSH (0.5 pgiml). GH (100 ngiml). SM-C (1 ngiml), retinoic acid (50 ngiml)

1 HC (50 nM), glucagon (0.2 pgiml), ascorbate (10 pgiml)

Key to Table II I =insulin; TF=transferrin; Clg=cold insoluble globulin (also known as fibronectin); EGF=epidermal growth factor; FGF=fibro- blast growth factor; TRH = thyroid releasing hormone; T3=triiodothyronine; PTH=parathyroid hormone; SM-C=somatomedin-C; HC = hydrocortisone; FSH =follicle stimulating hormone; LHRH=luteinizing hormone-releasing hormone; NGF=nerve growth factor; PGEl =prostaglandin-El; PGFz,,=prostaglandin-F z r i ; GH=growth hormone.

may play a vital role in cell interactions during the early stages of mammalian embryogene-

Therefore it was not surprising to ob- serve that fibronectin facilitates the attachment of several cell types in defined media (Table II, see also reference 62). While most studies suggest that fibronectin may play only a pas- sive role, the recent work of Orly and sat^^^ demonstrates that a non-tumorigenic rat ovary

cell line (RF-1) requires fibronectin in order to undergo cytokinesis in defined medium.

The role of insulin in defined media is cur- rently open to discussion and requires further study. Although insulin is needed by many cell types (Table II) it is required at concentrations (100 ng per milliliter or higher) that are much higher than those found in blood (1 to 4 ng per milliliter).63 This has led to speculation that

374 NUTRITION REVIEWSIVOL. 37, NO. 1PIDECEMBER 1979

Page 7: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

insulin, in at least some cases, may be sub- stituting for one of the somatomedins. Since insulin binds weakly to somatomedin receptors this would explain the need for concentrations 100 to 1000 times higher than cells are likely to be exposed to in vivo.e4>G5 It is not surpris- ing that insulin can bind to somatomedin re- ceptors since at least one somatomedin (IGF-1) shows a significant amount of amino acid homology with insulin.66 Alternatively, the need for high insulin levels may be the result of insulin’s rapid breakdown in defined media. Hayashi, Larner and sat^^^ have demonstrated that the insulin requirement for GH3 cells can be reduced approximately ten- fold if cysteine (which can reduce the disulfide bonds of insulin and destroy its activity) is re- placed with cystine. It is obvious that additional studies will be required in order to determine which hormones actually function in vivo.

In spite of such ambiguity, work with defined media is proving to be a powerful tool in many areas, three of which are discussed here: 1) the determination of the hormonal require- ments of normal cells, 2) the study of mechan- isms of hormone action and 3) the study of cell differentiation.

In regards to the first area, several members of this laboratory have recently cultured pri- mary tissues in hormone-supplemented de- fined media that had been developed for cell lines of comparable origin. These include func- tional pituitary, Sertoli, kidney epithelial and various neuronal ~ e l l s . 6 ~ - 6 ~ In each case the desired cell type was obtained without signifi- cant proliferation of fibroblasts, thus eliminating the most common problem of primary cultures. These studies demonstrate two important points: 1) defined media can be used to select for a given cell type, and 2) cell lines (even tumorigenic cell lines) can be used to predict the hormonal requirements of normal cells.

Defined media will also prove invaluable for the study of hormone action, as illustrated by some very recent data. Many hormones are known to interact with cells and initiate a number of responses, including a reduction in the number of their cell surface receptors (down regulation).70 In the past year it has been suggested that down regulation may be an im- portant step in generating the mitogenic signal

of EGF. Since studies demonstrating down regulation were conducted in serum-contain- ing media, Wolfe, Su and Sato recently decided to re-examine this question in defined me- dia.31-71 In direct contrast to the results ob- tained in serum-containing media, they have found that in defined media EGF does not cause down regulation of its receptor, yet still acts as a mitogen for HeLa cells.

Last, but not least, defined media can be used to identify the hormonal and nutritional requirements of cellular differentiation. Re- cently Rizzino and Crowley have demonstrated that the embryonal carcinoma cell line, Fg, can be grown in a defined medium (Table 11). When retinoic acid (a natural metabolite of vitamin A) is added to this medium, the cells undergo cellular differentiation.52 The cell type formed is similar to embryonic endoderm (parietal endoderm) in at least two respects: 1) morph- ology and 2) the secretion of plasminogen activator, the secretion of which is stimulated by dibutyryl cyclic adenosine monophosphate. This is the first demonstration of cellular dif- ferentiation in a hormone-supplemented de- fined medium.

Current and Future Perspectives In this section we discuss the interdepen-

dency of the nutritional and hormonal require- ments of cells, the nutritional and hormonal requirements of normal versus tumorigenic cells and the identification of the nutritional and hormonal requirements during the early stages of mammalian embryogenesis.

It has been accepted for some time that many hormones (for example, insulin and EGF) can affect the uptake of nutrient^.^^?^^-^^ It now appears that the nutrients present in the culture medium can influence the hormonal requirements of cells. Recently Wu and Sato75 have shown that HeLa cells grown in defined media require insulin and fibroblast growth factor (FGF) when the synthetic por- tion of the culture medium is F-12. In contrast, when Ham’s MCDB 105 (which contains nine more components) is used, the requirements for insulin and FGF are eliminated.75 This find- ing suggests that the hormonal requirements of HeLa cells can be influenced by the presence and/or the concentrations of nutrients. It pres-

NUTRITION REVIEWS,’VOL. 37, NO. 12iDECEMBER 1979 375

Page 8: Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture

ently is not known whether this is a general phenomenon and additional studies are necessary to resolve this question.

Turning now to the requirements of normal and tumorigenic cells, we believe that a com- parison of their nutritional and hormonal re- quirements would be fruitful. It is likely that such a comparison would provide important in- sights into the nature of growth control in vivo. In regards to hormones, it now appears that the requirements of normal and tumorigenic cells differ. There are two types of data which support this contention and also suggest how this area can be examined further. Firstly, several investigators have found that viral transformation of non-tumorigenic 3T3 cells leads to concomitant tumorigenicity and altered hormonal req~irements.~6-80 For example, when Balbi3T3 cells are transformed by SV40 their requirement for platelet-derived growth factor disappears.79 The second type of data has come from studies of cellular differ- entiation. Recently Rees et al.,al while examin- ing the differentiation of embryonal carcinoma cells to endoderm, observed that differentiation is accompanied by the appearance of func- tional EGF receptors ar;d an apparent loss of tumorigenicity. Thus both types of data sug- gest that hormonal requirements are relaxed when a cell becomes tumorigenic.

In regards to nutrients, it also appears that the requirements of normal and tumorigenic cells differ. Recent studies with a human cell line, WI-38, and its virally transformed counter- part suggest that some nutrients are needed in higher concentrations by the normal cells.38 The reason for this is unknown but it is tempting to speculate that alterations in the nutritional requirements (concentration dependence) are related to alterations in the hormonal require- ments. This seems reasonable since hormones are known to affect nutrient uptake. Perhaps a nutrient, whose uptake is controlled by a particular hormone in normal cells, can be taken up more readily by a cell that has become tumorigenic and no longer responds to that hormone. If the nutritional and hormonal re- quirements are interrelated as we have sug- gested, this raises a very important question. Did a change in the hormonal requirements or a change in the nutritional requirements

376 NUTRITION REVIEWSIVOL. 37, NO. IZIDECEMBER 1979

come first? Clearly, further study of this ques- tion is needed.

The nutritional and hormonal requirements of mammalian embryogenesis can also be ex- plored using defined media. For practical rea- sons it is difficult to formulate a defined medium for developing embryos using embryos di- rectly. In contrast, the model system of embry- onal carcinoma cells, which mimic early stages of mammalian embryogenesis can be used to predict the requirements of embryos. A s an example of this, Rizzino and Shermana2 have shown that partially purified fetuin, required by embryonal carcinoma cell lines in serum-free media,83 also supports the growth and differ- entiation of mouse blastocysts. While little is known about the nutritional requirements dur- ing embryogenesis, there is evidence that certain nutrients affect differentiation. For in- stance, it is known that the replacement of glucose with other sugars restricts the differen- tiation of embryonal carcinoma cells.84 A s in other areas that we have discussed, there is much work to be done in this field.

Summary We have stressed throughout this article

the importance of using defined media to probe the nutritional and hormonal require- ments of cells. We feel that this work would proceed most efficiently if the approaches used in this laboratory (to study hormones) and ha t of Ham (to study nutrients) are combined. As a result one could expect important theoretical (such as discovery of new hormones) and practical advances to be made in cell biology. As an example of the latter we predict that de- f ined media will replace the rout ine use of serum, just as serum replaced the use of embryo extracts more than 30 years ago. 0

The authors wish to thank Drs. W. Desrnond and D . Barnes for reading this review and making several helpful suggestions.

1.

2. 3.

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