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THE UTILISATION OF PROTEOSES BY CHICKENHEART FIBROBLASTS GROWING IN VITRO

BY E. N. WILLMER AND L. P. KENDAL.

(Physiological Laboratory, Cambridge.)

(Received $th October, 1931.)

(With Two Plates and Twelve Text-figures.)

CONTENTS.PAGE

Introduction ; 149Experimental methods

1. Preparation of media . . . . . . . . . . 152(a) Physiological saline solution 152(b) Embryo extract 152(c) Plasma 152(d) Proteose solutions 152

2. Preparation of cultures 1533. Measurements of growth in the cultures

(a) H a n g i n g d r o p c u l t u r e s . . . . . . . . . 1 5 4(b) Flask cultures 155

Experimental results

1. The effects of proteoses on growth 155

2. The effects of proteoses and plasma on growth 158

3. The effects of proteoses and serum on growth . . . . . . 1 5 8

4. The effects of proteoses, plasma and embryo extract on growth . . 160

5. A growth-promoting body in the heteroproteose fraction of Witte's peptone 165

Discussion 173

Summary 177

References 178

INTRODUCTION.

IT has been concluded by various workers, amongst whom may be mentionedLewis (1921), that native proteins are unsatisfactory as a source of nitrogen fortissues growing in hanging drop cultures; and Baker and Carrel (1926a), althoughthey consider that the main growth-promoting action of embryo juice is associatedwith the protein fraction, do not regard native proteins as capable of being utilisedas foods by the tissues.

It appears probable, although the evidence is not entirely satisfactory, thatJEBTXli JO

150 E. N. WILLMER and L. P. KENDAL

amino acids stimulate cells to increased activity for a short time when present inlow concentration (Ebeling, 1924; Baker and Carrel, 19266). In higher con-centrations they are toxic (Burrows and Neymann, 1917), and so far it has not beenpossible to keep cells alive for any length of time by adding amino acids to theusual physiological salt solutions used as culture media.

Following up their investigation of the properties of embryo tissue juice, Carreland Baker (1926) found that the higher fractions of peptic digests of proteins,namely the proteoses, were capable of producing a very marked increase in growthactivity when added to various tissues which were growing in plasma clots in"Carrel" flasks. Apart from embryo extract they are the only substances so fardiscovered which are capable of producing prolonged growth, and even they fallfar short of embryo extract in this respect. Yet these results, obtained in vitro, areof sufficient interest and importance to merit further investigation, and if theycould be shown to apply equally to tissues growing in vivo, new possibilities wouldbe opened up as to the mechanism of intermediate nitrogen metabolism in thebody. It must, however, be pointed out that the conditions which are suitable forproducing uncontrolled growth in vitro may not necessarily be identical with thosewhich support the cells in a healthy state in vivo. For example, cells like fibroblastsdo not show more than a very slight proliferative activity in the adult body, but intissue culture they can be made to grow at a rate at least as great as that of embryonictissues. On the other hand it is interesting to note that Fischer and Parker (1929)have described a method by which fibroblasts may be kept alive in a healthy,resting (i.e. not growing) condition for a considerable time outside the body. Theyachieved this result by frequently washing the cultures with heparin plasma, andit therefore becomes clear that the cells can obtain from the plasma alone sufficientnutriment for their existence in a resting condition, although they will not growextensively. The plasma therefore either does not contain those substances whichare necessary for cell multiplication, or alternatively it contains substances whichinhibit growth. Carrel and Ebeling (1921, 1923 a, 1923 b) and later Baker andCarrel (1925, 1927), by experiments on sera of animals of different ages, have shownthat both these possibilities are in some degree realised. They have been able toseparate from serum both inhibitory fractions and substances which promotegrowth; the former become more concentrated and the latter less so, as the age ofthe animal increases. The addition of proteoses, embryo extract, and certain other sub-stances can overcome these inhibitors, or supply the necessary stimulus for growth.

The immediate problems therefore are how the tissues obtain their nitrogensupply, what substances are necessary for cell multiplication, and what is the actionof proteoses and embryo extract when given alone, or together with plasma, as foodsubstances to tissues in physiological saline solutions. Is growth to be consideredas taking place automatically if the tissues are fully nourished, or are there specialsubstances which stimulate cell increase? The fact that cells can exist for greatlengths of time in the body without growing, and yet remain alive and healthy,suggests that a diet which is adequate for survival is not necessarily sufficient forgrowth. Mammals will not grow when lysine is omitted from the diet. The

Utilisation of Proteoses by Chicken Heart Fibroblasts 151

developing limbs of the frog depend on the activity of the thyroid secretion.Hammett and Reimann (1929) claim that wound healing is accelerated by theapplication of solutions of thioglucose. Such facts suggest that there exist certainsubstances which have a specific capacity for increasing the rate of multiplicationof cells. Is it then not merely a matter of degree whether these substances are to beregarded as foods in the true sense, or as growth stimulants? Foods are thosesubstances which must be ingested by the organism for its continued existence ina normal condition, and therefore a normal diet for resting tissue may requireadditional substances to render it adequate for growing tissue. It is not yet possibleto say whether these stimulants, if such they be, are actually built into the tissuesor whether their action resembles that of a catalyst. For a complete diet in vivo,vitamins are necessary, and some of these are definitely related to growth, butwhether they are to be regarded as foods or growth stimulants is undecided. Theyare called accessory food factors. In vitro, there are substances which definitelycause the cultures to increase in size and activity, and it was to investigate theproperties and mode of action of some of these substances, foods or stimulants,that the experiments to be described in this paper were undertaken. They arechiefly concerned with the action of proteoses and allied substances in promotinguncontrolled growth of chicken heart fibroblasts.

Carrel and Baker (1926), as already mentioned, have shown that fibroblastscultivated in a plasma clot in flasks are stimulated to increased growth activitywhen the supernatant fluid contains, in addition to the usual salts and glucosepresent in the Ringer-Tyrode solution, the products of the peptic digestion ofvarious proteins. This result has been confirmed by Fischer and Demuth (1927)and by the authors. Baker and Carrel (1928a, 1928*, 1928c) have also found thatnormal and sarcomatous fibroblasts respond differently to treatment with thesedigests. Whereas sarcomatous fibroblasts are able to proliferate for a considerabletime on peptic digests of certain pure proteins, e.g. crystalline egg albumen,edestin or casein, in normal fibroblasts this capacity is far less evident. Both typesproliferate freely for a time in digests of tissues and also of fibrin, but the digestsof purified proteins are only utilised by the sarcomatous cells, and in both casesthe media ultimately prove inadequate. For the continued growth of normal tissuesevery medium which has so far been investigated, with the exception of embryoextract, has failed to satisfy the requirements. It is uncertain whether the greatergrowth-promoting capacity of tissue or fibrin digests compared with that of purealbumen or edestin digests is explicable on the grounds of their more completeamino acid content or whether the former contain other substances which areabsent from the purified material. In any case the fact that normal cells cannotbe kept alive and growing indefinitely when digests of proteins are added to theplasma medium, together with the fact that when such digests are made from pureproteins they are almost completely ineffective in stimulating growth at all, suggeststhat something more than protein decomposition products is necessary for theprolonged growth of normal fibroblasts. Experiments to be described in this paperlend support to this hypothesis.

10-2


EXPERIMENTAL METHODS,

i. PREPARATION OF MEDIA.

(a) Physiological saline solution. The modified Tyrode solution used throughoutthese experiments has been made up in glass-distilled water according to thefollowing formula:

NaCl o-8 % NaH2PO< 0-005 %KC1 0-02 % MgCl* o-oi %CaCl2 0-02 % Glucose o-i %NaHCOs 0-05 %

In the earlier experiments the ingredients were made up in separate solutionswhich were then sterilised in steam, and mixed in the requisite proportionsimmediately before use. Latterly the solution has been made up as a whole andsterilised by filtration through a Berkefeld candle. The amount of NaHCO8 inthe earlier experiments was o-i per cent., but this was found to render the solutiontoo alkaline and was consequently reduced later so that the pH of the final solutionshould be 7-5. A litre has generally been made up at a time, and after nitrationhas been stored in sterile Pyrex tubes in a refrigerator until required for use.

(b) Embryo extract. Chick embryos of 9-12 days' incubation are extracted fromthe eggs under strictly aseptic conditions: the eyes are removed, and the rest of thetissue washed as free from blood as possible by frequent rinsing with Tyrodesolution. They are then placed in centrifuge tubes and ground to a pulp by meansof a glass rod. To this pulp is then added 5 c.c. of Tyrode for each embryo, and thewhole is thoroughly mixed and allowed to stand for several minutes before beingcentrifuged (2000 rev. per min. for \ hour). When this operation is complete thesupernatant fluid is used at once. It generally contains about 60 mg. nitrogen per100 c.c. Its pH is about 7*3.

(c) Plasma. Blood is obtained from an adult fowl by bleeding from the carotidartery in the manner described by Strangeways (1924). It is then centrifuged atonce, and the plasma pipetted off into waxed tubes. These are stored in a re-frigerator till required. The whole operation is carried out under strictly asepticconditions.

(d) Proteose solutions. The proteo3es have, except where otherwise stated, beenobtained in the following way. A 20 per cent, solution of Witte's peptone in distilledwater is prepared, the commercial product being in such proportions almostcompletely soluble. The dark-coloured, somewhat viscous solution is then pouredinto four times its volume of distilled water, which causes the formation of a whiteflocculent precipitate amounting to 3-5 per cent, of the whole "peptone." Thesolubility of this substance in the concentrated solution seems to be dependent ontraces of salts, contained originally in the Witte's peptone, and probably on thepresence of a high concentration of the protein cleavage products themselves. Ondilution both these stabilising factors are rendered ineffective and the precipitate isformed. It is removed by centrifuging and its effects on the growth of tissues

Utilisation of Proteases by Chicken Heart Fibroblasts 153

in vitro will be described later in this paper (substance X). A strong solution oftrichloracetic acid is added so as to bring the concentration of the reagent in themixture to 2-5 per cent. After standing for several hours the precipitate is removedby the centrifuge or by filtration, and the clear fluid is heated on the water bathuntil the smell of chloroform can no longer be detected, and the hydrogen-ionconcentration has returned to that of neutrality. This removal of trichloracetic acidis a tedious process and when the volume of the solution is of the order of 1-2 litresit may take 2-3 days. Distilled water is added from time to time to maintain theoriginal volume. The trichloracetic acid-free solution is saturated with sodiumsulphate at 38° C, and after standing for about half an hour at this temperature,the sticky mass of precipitate is washed with saturated Na,SO4 solution, and re-dissolved in a volume of distilled water equal to that of the original solution. Thisprecipitation with NagSOf is repeated three times. Finally 10 per cent, bariumchloride solution is added in just sufficient quantity to precipitate all the sulphateion present; excess of barium chloride is avoided. The solution is filtered anddialysed in collodion sacks, first against running tap water and then against distilledwater, until free from the chloride ion. To the dialysed solution, twice its volumeof alcohol is added, and the precipitated proteoses separated by centrifuging. Bywashing successively with alcohol and ether, and removing the last traces of thelatter by evaporation in vacuo over concentrated sulphuric acid, a dry product isobtained. This consists of those substances usually classed together as a proteoses,and these have been shown by Carrel not to differ in activity from the j3 proteoseswhich are not precipitated by 66 per cent, alcohol. The product can be kept in thedry state and weighed out as and when required. In many of the more recentexperiments the method of preparation has been somewhat different, as will bedescribed in connection with those experiments where such preparations havebeen used.

2. PREPARATION OF CULTURES.

The tissue throughout these experiments has been taken from the ventricle of theheart of the 10-day chick embryo. Circumstances have sometimes necessitatedthe use of slightly older or younger chicks, but in no case has the age exceeded12 or been less than 9 days. The use of a pure culture of fibroblasts was not at firstpracticable, so that for the sake of uniformity fresh tissue has been used throughout.

The cultures were made in hanging drops, or by means of the flask technique, inwhich tissues are grown in a solid clot consisting essentially of plasma, above whichis a fluid phase. The latter can be frequently renewed and varied at will. The flasksused have been of the Carrel and Ebeling Type A (1923 c), 3-5 cm. in diameter, andtwo fragments of tissue have been planted in each flask. All glassware has beenthoroughly cleaned by the use of chromic acid cleaning mixture followed by severalwashings with distilled water. Sterilisation has been effected by dry heat at 1450 C.for 1 hour. For hanging drop cultures it is absolutely essential that the coverslipsbe entirely clean and free from grease, otherwise irregular results follow. To becertain of their complete cleanliness each coverslip has been treated separately with


5 per cent. NaOH, water, strong chromic acid mixture, and frequent changes ofdistilled water. They have been kept in alcohol until required for use.

In putting up the cultures in flasks the volume of the clot has been i c.c.throughout the experiments, which gives a depth of just under i mm. Experimentsupon the effect on the area of growth of altering the depth of the clot showed thatif the thickness was increased much over i mm. the growth became less extensive,more compact, and the cells became fatty in appearance. In the experiments inwhich this point was demonstrated, the medium was made up of constant pro-portions of plasma and embryo extract, and it at once became clear that the areaof growth depended on the depth of the tissue below the surface. Previous workersalso have shown that the density of the plasma clot has a marked influence on theresulting area of growth, so that in all the experiments described in this paper greatcare has been taken to ensure that the clot has always been of the same depth andits constituents present in the same proportions, except where alterations have beenmade for a definite purpose, in which case specific mention is made as to theeffects produced.

3. MEASUREMENT OF GROWTH IN THE CULTURES.

(a) Hanging drop cultures. Two methods have been employed.Method I. The cultures have been examined daily and numbers have been

assigned to each according to the amount of new growth observed: thus, culturesgrowing poorly have been given one, and those showing more growth, highernumbers up to four. This method has been fully described in a previous paper(1927), and although very arbitrary, has the advantage that by its use some allowancecan be made for the density as well as the extent of the growth, and the behaviourof the tissues can be observed for several days.

Method II. The cultures have been fixed (Bouin's fluid) and stained (haemalum)after they have been incubated at 390 C. for more than 24, but not more than 40hours. The areas of new growth have been drawn on squared paper by means ofa camera lucida, and the mitotic figures present counted by systematically examiningthe field of new growth under a f in. oil immersion lens. Photographic records ofgrowing cultures made by means of a modified cinematograph apparatus haveconfirmed the conclusions reached by Spear (1931) that cultures grow uniformlyduring the period mentioned, and that in suitable media mitoses are then offrequent and regular occurrence. If cultures are fixed at an earlier age than 24 hoursthe extent of growth is small and rather irregular, since the explants do not all atfirst adhere equally to the glass and may suffer more or less injury in the makingof the cultures. After 40 hours, hanging drop cultures tend to degenerate, and boththe counts made by Spear and the photographic records show a decrease infrequency of cell division.

The examination of fixed and stained cultures gives an insight into the characterof growth and the degree of activity of the tissues at a given moment, which,although thus limited, when taken in conjunction with Method I gives valuableinformation. It is important to know to what extent the growth of a culture is due


to cell division, rather than to a mere outwandering of the cells from the originalimplant: and also to judge by an examination of the cells whether they are largeand healthy, or small and emaciated, vacuolated, or loaded with fat. Sometimesthe cells are closely packed together, and in other media they become widely separatedand scattered over a large area. In consequence of these considerations, greatcaution must be observed in drawing conclusions from the results obtained by anyone method, and a true conception of the condition of the culture can only beobtained by a synthesis of the combined data.

An attempt has been made by means of continuous photographic records ofgrowing cultures to estimate how far the new growth in a culture is made up of cellswhich have migrated directly out from the implant, and how far it is made up ofthose which have arisen by division. The results of the preliminary experimentsusing fresh embryonic tissues have not been very consistent, but in cultures growingin embryo extract it has been estimated that in any given period between 20 and40 hours' cultivation the number of new cells in the field produced by mitosis formsabout 40 per cent, of the total increase in cells. Further experiments are, however,necessary on a strain of tissue more uniform in its growth rate than fragments ofchicken heart, but this result serves to illustrate how important a part the migrationof cells plays in the formation of the area of new'' growth,'' and what great variationsin area are to be expected by alterations in the migratory activity of the cells.

(b) Flask cultures. In flask cultures the area of new growth has been estimateddaily by making camera lucida drawings on centimetre-squared paper with amagnification of 21 diameters, and counting the squares covered. The average ofthe actual sizes (in area) of the cultures has been taken for those growing in anygiven medium, and the figures so obtained directly plotted against the age of thecultures.

EXPERIMENTAL RESULTS.

1. THE EFFECTS OF PROTEOSES ON GROWTH.

If proteoses are to be regarded as the immediate source of nitrogen for thetissues it might be expected that the growth of tissues in hanging drops of salinemedia would be enhanced by the addition of proteoses in the same way as theiractivity is increased, for example by the addition of glucose. Dialysates of embryoextract have been found to stimulate the tissues under such conditions, and thisis probably to be regarded as being due to their content of amino acids, since thesein low concentrations have been found to produce similar results. But as em-phasised by Baker and Carrel (1926 b), such solutions only act as temporary stimulantsand do not allow of indefinitely prolonged growth. In the light therefore of thesuccessful results obtained by Carrel and his school on adding proteoses to tissuesin plasma clots, it was decided to add proteoses, prepared in the manner describedabove, in various concentrations to a medium consisting of Tyrode solution alonein order to see if they were directly utilised by the cells in hanging drops in theabsence of plasma. Cultures were made in solutions in which the nitrogen con-


centration varied from 16 mg. to 170 mg. per 100 c.c. The proteoses were dissolved

2-0

1-8

1-6

| 1-2

I| 0-8

0-6

0-4

0-2

0 1 2Time in days

Fig. 1.

32 mg16 mg

65 mg.

Tyrode130 mg

19 mg

1 2 3 4Time in days

Fig. 2.Figs. 1 and 2. Effect of purified proteoses on growth of chick fibroblasts in hanging drop culture.The concentrations of proteoses are expressed in mg. nitrogen per ioo c.c. of medium. Growthestimated by Method I (see p. 154).

in distilled water, steam sterilised, and then treated with an equal quantity of doublestrength Tyrode solution. The results are shown in Text-figs. 1 and 2 and also in


Table I. The preparations A and B of the proteoses used in the experiments fromwhich the table was derived were made in a manner to be described later (p. 172).Preparation B is comparable to the preparations used for the experiments illustratedgraphically (Text-figs. 1 and 2).

Table I.

Numberof exp.

1

2

3

Durationof growth

(hours)

30

4 0

40

Medium

Proteoses:Preparation A

»»»»

TyrodeProteoses:

Preparation B

>)TyrodeProteoses:

Preparation A>>

Preparation B

Tyrode

Nitrogenconcentra-tion (mg. Nper 100 c.c.)

804 02 01 0—

56196

—

17030

170

3°

Numberof cultures

99999

8999

1 01 08

1 01 0

Averagearea of

new growthper culture

5-i5-24'33-2i-o

6 04'94 02 ' 0

S-9io-o3 97'45'9

Averagemitoses

per culture

i-o3'31-91 90-2

2-01 91-30 9

1-22 90 4OS1 6

These experiments clearly showed that proteoses, in the concentrations usedby Carrel and Baker in their flask cultures, are of no value to cells growing inhanging drops of Tyrode solution. In very much lower concentrations a small butdefinite activity was observed, but in no case was the growth comparable to thatobtained in embryo extract, or even in dialysates of embryo extract which in hangingdrop cultures produce results comparable with those obtained with the wholeextract (Wright, 1926a). These experiments therefore clearly indicate the prob-ability that the proteoses themselves, although utilised to a small degree if sufficientlydilute, cannot directly satisfy the nitrogen requirements of chick heart fibroblasts.Similar experiments were repeated in flasks without any form of coagulum usingthe method suggested by Fischer (1930a), and the results were again negative, atleast when the nitrogen concentrations were of the order of 170 mg. N per 100 c.c.

Ox fibrin was also digested by pepsin and HC1 and the products of digestiontested on hanging drop cultures. 5 c.c. or 10 c.c. of Benger's pepsin were added to200 c.c. of iV/20 HC1 containing 20 gm. of washed fibrin and digestion carried onfor 2, 4 or 6 hours. The resulting digests were neutralised by NaOH, boiled andfiltered, and then dialysed to free them from salt. During the dialysis a copiouswhite precipitate settled out; this was filtered off, and the filtrates after making upto the requisite salt and hydrogen-ion concentrations (/>H 7-5) were tested onhanging drop cultures with similar negative results.


2. THE EFFECTS OF PROTEOSES AND PLASMA ON GROWTH.

With these results in mind it was decided to use the proteoses in flask culturesin the way described by Carrel and Baker in order to verify their activity. Cultureswere made with different concentrations of proteoses in the fluid phase, and thesolid phase was composed entirely of plasma, or plasma diluted with Tyrode, andwas allowed to clot spontaneously in the incubator at 390 C. without the use ofany embryo extract. Under such conditions the coagulum is slow in forming andtends to become rather easily detached from the glass, but owing to the greatgrowth-promoting activity displayed by embryo extract it was thought advisableto eliminate it if possible in" testing the nutritive value of the proteoses. The resultsobtained (Text-fig. 3) were satisfactory in showing that the proteoses now producedan undoubted increase in the growth of the tissues, and that solutions which inhanging drops were valueless or even slightly inhibitory to growth, producedresults similar but markedly inferior to those reported by Carrel and Baker; that

60r

50

I 40!so

20

10

bo

1 °/o Proteoie

0-1 °/o ProteoseTyrode0 01 °/o Proteose

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Time in hours

Fig. 3. Growth of 11-day chick heart fibroblasta in proteose solutions in flask cultures.

is to say,solutions up to 2 per cent, showed increasing activity. For this there appearto be two possible explanations: either the plasma clot acts in a protective capacityto the tissues, or it aids in some way in rendering the proteoses utilisable by thetissues, supplying either an enzyme, or perhaps a co-enzyme to an enzyme presentin the tissues, the system thus providing the cells with a constant source ofamino nitrogen in low concentration and at the same time protecting them fromthe harmful effect of the strong concentrations of the proteoses. Carrel and Baker(1926) have suggested that the activity of the proteoses is dependent on their slowdigestion by the tissue enzymes, but it appears from the above experiments thatthe plasma coagulum plays some important part in this digestion, unless its actionis merely protective.

3. THE EFFECTS OF PROTEOSES AND SERUM ON GROWTH.

With the idea of being able to eliminate the mechanical factors introduced bythe plasma clot, cultures were made in which the plasma was replaced by serum.The serum was obtained from an adult fowl by allowing the blood to clot spon-


taneously at 390 C , separating the clot by means of a knife from the containingtube and allowing it to contract. For this purpose it was left in the incubator forabout 12 hours. The serum when expressed from the clot was pipetted off andcentrifuged. Cultures were made both by the hanging drop method and in flaskswithout plasma after the manner described by Fischer (1930ft). The serum soobtained was not inhibitory when diluted with an equal volume of Tyrode, butalways caused the appearance of quite considerable areas of growth, which weremuch greater than those obtained in Tyrode solution alone (PI. I, fig. 1). Countsof the number of mitoses occurring in serum clearly indicated that at least some ofthe area of new growth was due to cell division. The cultures tended to spread outregularly at first, but in many cases a break occurred in the new growth and muchof it retracted on to the central implant, leaving a ring of new cells isolated at somedistance out in the field. This naturally made measurements of the amount of growthrather unsatisfactory and irregular.

Table II.

OU1UL1UI1

Serum and proteoses(80 mg. N)

Serum and Tyrode

Proteoses (80 mg. N)and Tyrode

Tyrode

ptl

7 6

7-8

7-S

7'9

Measurements of cultures, fixed andstained 34 hours after planting

Area 17,32,16,12,17,19,15,17,17Mitoses 37, 261, 125, 30, 119, 99, 102, 82, 82Area 20,28,27,18,18,28,31,28,18,20Mitoses 71, 50,47, 57,43,71,46, 195, 28,49Area 7,6,9,11,4,6,6,8,6,3Mitoses 2 , 1 , 2 , 1 , 0 , 2 , 0 , 1 , 0 , 0Area 1 , 6 , 5 , 6 , 7 , 3 , 3 , 6 , 4 , 4Mitoses 0 , 3 , 1 , 4 , 0 , 1 , 0 , 0 , 0 , 0

Averagearea

18

24

6 6

4'7

Averagemitoses

perculture

1 0 4

66

0 9

0 9

Averagemitosesper unit

area

s-s

2-8

o - i

O-2

When an equal volume of proteose solution, containing 170 mg. N per 100 c.c,was added to the serum the areas of growth were practically unaffected. Thegrowth was, however, rather thicker, and the tendency for the central fragment toretract, and so detach much of the new growth, was even more marked than in serumand Tyrode mixtures (PI. I, fig. 2). Counts of the mitoses in the new growthshowed much greater numbers in the presence of proteoses, so that it is evident thatthe cells were making use of the proteoses under the influence of the serum, while thesame proteose solution, when added to Tyrode, produced no beneficial results, or onlya slight increase in area. Table II shows the result of an experiment illustrating thispoint. These cultures were fixed 34 hours after planting. Although the differencesin pH of the media are perhaps rather large, they are not sufficient in themselvesto produce significant changes in growth in such an experiment. Of the culturesgrowing in the serum plus proteose medium eight out of nine showed retraction ofthe new cells from one side of the culture on to the central fragment, while of thosein serum alone only one showed any tendency in this direction. The breaking awayof the implant from the new growth is probably correlated with the rhythmiccontractions of the implant, since these were noticeably greater in the cultures in

i 6o E. N. WILLMER and L. P. KENDAL

proteoses plus serum, and continued in these solutions for longer than in serumalone. This observation adds confirmation to the results of the experimentsdescribed above, in that it suggests that the proteoses were being utilised by thetissues as a food supply. Another experiment in which the cultures were fixed after48 hours showed the serum cultures with few mitoses and starting to degenerate,whilst the growth with proteoses present was noticeably thicker and mitoses werestill abundant. Table III shows the figures for the best healthy cultures in eachmedium.

Table I I I . Numbers of mitoses in surviving cultures after 48 hours' growth.

Solution

Number of mitosesin each culture

Average

Proteoseaand serum

69

%33

56

Serumand Tyrode

90212

2-8

Proteosesand Tyrode

322

31

2-2

Tyrode

00

0

The results obtained with serum in the medium were rather irregular, andserum freshly prepared gave better results than when it had been standing for anylength of time, even at o° C. The areas of growth in serum alone were large, sothat the extra effect of the proteoses is correspondingly difficult to detect, but it ismade evident by the numbers of cell divisions. There is no doubt that in some way,probably by the action of digestive enzymes, serum renders proteoses available tothe tissues.

It is possible that some of the growth-promoting effects of the serum areconsequent on its method of preparation in which it remains in contact with theblood cells at 390 C. for several hours, during which time metabolites probablyaccumulate and the leucocytes may produce those substances (trephones) whichwere found by Carrel and Ebeling (1922) and Carrel (1924) to activate the growthof fibroblasts. But the fact that both plasma, which is immediately separated fromthe corpuscles, and serum render the proteoses available to the tissues suggeststhat there is in both an enzyme which brings about this result.

Attempts were made also to culture tissues in serum and proteoses in flaskswithout any solid coagulum, but the results were unsatisfactory, as the tissuesretracted and remained completely rounded off. The mechanical properties of theplasma coagulum are evidently very important in the ordinary flask cultures. Anattempt was made to see how far these were responsible by using heparin plasma,instead of serum, but the heparin was insufficient to prevent the formation of clotsin the immediate vicinity of the tissues.

4. THE EFFECTS OF PROTEOSES, PLASMA AND EMBRYO EXTRACT ON GROWTH.

It was reported earlier in this paper that the growth obtained in flasks withplasma as the solid phase and proteoses in the fluid phase was considerably greaterthan that obtained with Tyrode as the fluid phase, but that the results were not as


satisfactory as those described by Baker and Carrel. The cause of this differencepossibly lay in the fact that embryo extract had been omitted from the constitutionof the clot, whereas the coagula used by Baker and Carrel always contained aproportion of extract for the purpose of causing quicker and more completecoagulation. Experiments were therefore carried out with different concentrationsof embryo extract in the coagulum. The clots were covered by a fluid mediumconsisting of a i per cent, solution of proteoses in Tyrode solution, or by Tyrodealone as controls.

Table IV.

Quantityof embryoextract inthe clot

Nil

Nil

o-i c.c.

o-i c.c.

0-2 C.C.

0-2 C.C.

Fluid mediumadded

i % proteose inTyrode i c.c.

Means

Tyrode I c.c.

Means

i % proteose inTyrode i c.c.

Means

Tyrode i c.c.

Means

i % proteo9e inTyrode i c.c.

Means

Tyrode i c.c.

Means

Area of cultures

o

I"2i - i0-9

I - I

0 9i-7i-6i-4i'4

I-I

i-6i"3i-3

i'3

I-I1 3i'3I-I

1-2

I-Oi 6i-7i-o

i'3

I-Ii'309i 41-2

22 hr.

S-37'34-2

S-6

i-7

s-s43433-9

9-2II-210590

io-o

34395'549

4-4

140IS S!6-SII-O

14-2

5-i6-5508-362

46 hr.

n-6iS-SI I - I

127

5'oio-o7-i6-S

7-i

2221271922

7'5

908-i

42454528

40

12-SI2-O12-0130

124

70 hr.

15-521

18-2

IO-215-0io-8io-o

II-S

30303327

30

n-5II-O130

12-S

I2-O

60636l40

S6

1716-519i8-518

94 hr.

2026

23

12IS1110

12

374°3630

36

IS12ISIS

14

7180805872

222O2424

23

The cultures were made in Carrel flasks in the manner already described, andthe result of a typical experiment is shown in Table IV, together with details as tothe variables in the composition of the media. The table shows the areas of thecultures from day to day, traced on squared paper by camera lucida drawings, andon comparing the different sets of cultures it is seen that where the fluid medium


contains i per cent, proteoses the increased growth produced by a small quantityof embryo tissue juice in the clot is much greater than that produced when the fluidmedium consists of Tyrode solution alone. By dividing the average areas attainedwith a proteose fluid medium by those attained in the same time with a Tyrodefluid medium, the ratio of the areas in proteose solution to those in Tyrode solution,termed by Carrel the growth index of the experimental medium, is obtained. Theseindices are shown in Table V, and are definitely greater when the clot containsextract than when it does not, and still greater when the clot contains o-2 c.c.embryo extract than when it contains only o-i c.c.

Table V.

Quantity ofembryo extract

in the clot

NilO'l C.C.O 2 C.C.

22 hr.

i-42323

Area in proteose mediumArea in Tyrode

46 hr.

182'73-2

70 hr.

162-53 1

94 hr.

192 63-i

A number of other experiments on the same lines as the above were carriedout, and, as they gave the same general result, they need not be described in detail.The effect of embryo extract on the utilisation of proteoses is seen even moreclearly when the average figures obtained in the whole series of experiments aretabulated together in groups, each group comprising the average areas on successivedays of sets of cultures grown with the same fluid medium and the same amountof embryo extract in the clot. The collected data are set down in Table VI andshown graphically in Text-figs. 4 and 5.

Table VI.

Solid phase

No extract

c i c.c. extract

o#2 c.c. extract

0-5 c.c. extract

Fluid phase

Tyrode1 % proteosesTyrode1 % proteosesTyrode1 % proteosesTyrode1 % proteoses

1st day

1 6

1'417

M11

2nd day

6-s±i-7

6 - I ± I - 7

7-o±o6I2'4±2'2

3rd day

65 ± 1 214-0 ±38-6±2-6

24-0 ±512 5±i 6

17 ± 433 ±7

4th day

8-6± i-6i7-o± 2I 2 O ± 2-4

38o± 8170+j 2050-0 ±14

29 ± 373 ±14

Sth day

io-6± 2-323'O± 2i6-o± 3-1S9-o±i624-0 ± 2700 ±15

S3 ± 9134 ±18

NJ3. The 1st day measurements were made when the fluid phase was added, approximately16 hours after implantation; hence the tendency for the initial areas to increase with greater amountsof extract in the clot.

These experiments clearly show that the presence of small quantities of embryoextract has a very great effect on the growth of cells in proteose solutions, and theincreased growth which then appears is much greater than would be expected ifproteoses and extract acted independently, and if the combined effect of the twowere merely the sum of their separate effects. There are several possible explanationsof this behaviour. In the first place the mechanical properties of the clot are altered

Utilisation of Proteoses by Chicken Heart Ftbroblasts 163

by extract. The clot is much firmer, and probably contains smaller meshes owingto its quicker coagulation. This is probably not the cause of the increased growth,since Lambert (1914) has concluded from the beneficial effects on growth ofdiluting the plasma with saline solutions, which would lead to clots with largemeshes, that coarseness and not firmness of the clot favours growth. Secondly, theextract may change the metabolism of the cells in a Way which enables them moreeasily to utilise the proteoses, i.e. it may act directly on the cells; or thirdly, it mayact on the proteoses and hydrolyse them to a more readily accessible form. Demuth

140

130

120

110

100

1 90

1 80o§ 70«S, 60S

ij 5040

30

20

10

1 7.

Fluid mediumProteose in Tyrode solution

0 5 c cEmbryo extract

o 25 c.cEmbryo extract

0-1 ccEmbryo extract

Without extract

0 1 2 3 4Time in days

Fig. 4. Growth of chick heart fibroblasts in plasma clots containingvarying quantities of embryo extract.

and von Riesen (1928), who examined the digestion occurring in various media byfollowing the change in the amount of nitrogen which could not be precipitatedby phosphotungstic acid (according to the method of Folin and Wu), found thatembryo extract has a marked capacity for hydrolysing proteoses added to it, andthis result has been confirmed by the present authors in the following way. A measureof the amount of digestion occurring during a period of incubation of various mediawas obtained by determinations of the amino nitrogen content at the beginningand end of the period. For the determinations the micro-manometric method ofVan Slyke (1929) was used, and the media were made up aseptically and containedin sterile stoppered pyrex tubes, so that it was unnecessary to add any bacteriocide.


When plasma was used, it was treated with heparin in order to prevent clotting.The media were incubated at 39° C. for a period of 4 days. Table VII gives thedetails of one of these experiments. The figures in the last horizontal row indicatethe percentage increase in amino nitrogen which must be ascribed to the proteoses

00

Clll

tl

o

s3V

2<

140

130

120

110

100

90

80

70

60

50

40

30

20

10

Fluid mediumTyrode solution

-

•

-

-

-

•

-

y

^ ^ ^ - ^

o 5 c c/ Embryo extract

//

0-25 c c^_^-» Embryo extract

o-i t c^ "* Embryo extract— • Without extract


Fig. 5. Growth of chick heart fibroblasta in plasma clots containingvarying quantities of embryo extract.

in the mixture, allowance having been made for that due to autolysis in the plasmaor extract itself.

Table VII.

Plasma (cc.)Embryo extract (cc.)Solution of proteoses in o-8 % NaCl (cc.)Tyrode (cc)Mg. amino N per ioo cc.:

Initial ... ... ...After 4 days ... ...

Percentage increase in amino N in 4 daysPercentage increase in amino N in 4 days

due to digestion of proteoses ...

1

6-5

x8-5

8-53946

10-9

2

12-5

125

4'435'4i

22-2

3

6-5

3-S150

22-1

43-898-2

152

4

12-53-S90

17-226-6

547

66


As in the experiments of Demuth and von Riesen (1928) the figures show thatplasma digests the proteoses much more readily than does embryo extract, and itis therefore unlikely that the effect of small additions of extract on the growth oftissues in plasma and proteose media is dependent on the proteolytic activity of theextract, although there is still the possibility that the course and end products ofdigestion brought about by the enzymes of the extract may be different from thoseresulting from the breakdown of proteoses in plasma. It is more reasonable tosuppose that the extract supplies some factor which by its beneficial effect on thegeneral cell metabolism enables the cells to utilise the available nitrogenous sub-stances more effectively. In the above experiments the digestive activity of serumwas found to be of the same order as that of plasma.

Gosely connected with the question of proteolytic enzymes is the liquefactionof the coagulum which frequently occurs in the immediate vicinity of the tissues,and which has been described by Mayer (1930). If embryo extract is left in contactwith a plasma clot at a temperature of 390 C. for a sufficiently long time the latteris gradually eaten away, but this process is slow and probably rather different incharacter from the sudden liquefaction which is associated with the presence oftissues. Cultures which on one day appear perfectly healthy and actively growing,on the next may be reduced to a thin ring of, tissue surrounding a large circularcavity in the coagulum. This liquefaction and sudden retraction has been par-ticularly noticeable in cultures growing in media containing proteoses, although itis by no means confined to these. Demuth and von Riesen, who estimated theamount of nitrogen in the fluid phase of cultures which was not precipitable byphosphotungstic acid, found that this quantity did not increase after the lique-faction, so that if the latter is due to digestion the process does not proceed very far,unless the proteolytic products are absorbed by the tissue. At present the causeand significance of the liquefaction is obscure. The clot is manifestly weakenednear the tissue, and when strained by contraction of the tissue, or by changes intemperature following removal from the incubator, it gives way, and a circular rentappears.

5. A GROWTH-PROMOTING BODY IN THE HETEROPROTEOSE FRACTION

OF WITTE'S PEPTONE.

During the preparation of the pure proteoses, which has already been described,a precipitate formed during the first dilution with distilled water of the 20 per cent,solution of Witte's peptone, and a similar substance appeared on treating pepticdigests of fibrin in the same way. If during the preparation of pure proteoses theconcentration of these latter in the solution was maintained high, i.e. of the orderof 10 to 20 per cent., then a precipitate appeared on dialysing the final solution torender it free from salt. These precipitates were all partially soluble in salt solutions,and when an extract of them in Tyrode solution was used as a culture medium forembryonic chick heart tissue a large area of growth and numerous mitoses resulted.It was found most convenient to prepare such an active solution by the first of thethree methods mentioned above. When the 20 per cent, solution of Witte's peptone

JEB'IX ii U


is diluted ten times with distilled water, about 4-5 per cent, of the nitrogenousmaterial is precipitated. Most of this is found to be insoluble in neutral saltsolutions, cannot be utilised by tissues in vitro, and may be considered to be meta-protein. If the precipitate is washed by repeated suspension in a large volume ofdistilled water, the nitrogen content of successive washing fluids decreases until itbecomes constant at about 1 mg. per 100 c.c, and the washings display no activitywhen tested as culture media. If now the insoluble material be extracted witho-8 per cent. NaCl solution the extract is found to contain, in addition to the1 mg. per 100 c.c. of N which would be dissolved by distilled water, a furtherquantity of dissolved nitrogen, the amount present being independent of thevolume of extracting solution, but definitely related to the original amount ofWitte's peptone taken; 100 gm. of Witte's peptone yield roughly 30 mg. N in thisway. Further extraction of the residue with o-8 per cent. NaCl shows a return of theN content of the extracting solution to 1 mg. per 100 c.c, and it is in the firsto-8 per cent. NaCl extract only that the growth-stimulating substance is found.These facts suggest that the active agent is a nitrogenous substance, and indeed ithas not been possible to obtain an active solution of it which has not given a well-marked biuret reaction. Its solubility properties are those of a heteroproteose, andit is quite stable in neutral solution at 100° C.

The method actually employed for obtaining a solution containing this activeagent is as follows. 20 gm. of Witte's peptone are dissolved in 80 c.c. of distilledwater, the solution filtered through muslin and made up to a litre with distilledwater. A white flocculent precipitate is formed, and after standing for several hoursis separated by centrifuging. It is washed twice by suspending in 300 c.c. ofdistilled water, and finally extracted with 50 c.c. of o-8 per cent. NaCl solution.This solution contains the active substance, and after removing the remaininginsoluble material by means of the centrifuge, the solution is sterilised in steam. Foruse as a culture medium it is added to an equal volume of a solution containingo-8 per cent. NaCl and the other constituents of Tyrode solution in twice the normalconcentration, so that the final solution contains salts and glucose in the samequantity as they are present in normal Tyrode. For convenience in describing theexperiments the growth-stimulating substance obtained in the above way will bereferred to as substance X. In hanging drop cultures a medium containing sub-stance X determined a large area of outgrowth of cells which commenced toemigrate at once from the explant (PI. I, fig. 3). Most often the cells spread outuniformly and rapidly in a single layer on the surface of the coverslip (PI. I,fig. 4). The cultures also very often showed cells migrating in a single layer on thefluid-air interface, but during fixation this layer is usually lost, so that the actualareas shown in the accompanying tables do not do full justice to the amount ofgrowth. Table VIII shows the areas of growth in a solution containing substance X,the nitrogen content of the undiluted medium being 18 mg. per 100 c.c. Thecultures were fixed after 48 hours. Table IX shows the results from a similarexperiment in which the cultures were fixed after 37 hours' growth, and gives alsothe numbers of mitoses present in the area of new tissue. Text-fig. 6 shows in


graphical form the relationship between the concentration of substance X and theamount of growth obtained, which may be compared with Text-figs. 1 and 2 whichshow similar curves for solutions of pure proteoses, and it is at once evident thatsubstance X produces a much greater growth.

2»6

2-4

2-2

2-0

1-8

I"6

I 1*4

a 1-2

< 1-0

0-8

0-6

0-4

0-2

20-6 mg N

10-3 mg N5 15 mg N

2 57 mg N

Tyrode


Fig. 6. Growth of chick heart fibroblasts in different concentrations of substance X. The con-centrations are expressed in mg. nitrogen per ioo c.c. of solution. Nine-day old chick. Growthestimated according to Method I (see p. 154).

Table VIII.

Solution

Substance X 18 mg. N per 100 c.c.Substance X 3-6 mg. N per 100 c.c.Tyrode

Measurements of area of growth.Cultures fixed and stained after 48 hr.

25, 32, 21, 29, 36, 27, 33, 26, 391 1 , 1 3 , 1 8 , 1 5 , 2 0 , i i , 171,3,8,4, i , 5 , 7 , 7

Averagearea

3O±5iS±34 ± 3

The cells growing in solutions containing substance X tend to spread out in asingle layer and often become fairly widely separated (PI. II, fig. 1). Histologicallythey appear somewhat emaciated, with very pronounced processes. Mitoses arefrequent, and often occur in cells which, at least when fixed and stained, arecompletely isolated from their neighbours (PI. II, fig. 2). After prolonged growththe cytoplasm becomes small in proportion to the size of the nucleus, which oftenassumes an irregular shape. After fixation and staining many cells in these cultures

11-2


show nuclear patterns resembling the prophase of mitosis. This, however, appearsto be a degenerative phenomenon and only occurs in old cultures in which normalmitoses are rare or absent (PI. II, figs. 3 and 4).

Table IX.

Solution

Substance X 18 mg. Nper 100 c.c.

Substance X 3 mg. Nper 100 c.c.

Tyrode

Measurements on cultures fixed andstained after 37 hours' growth

Area 17, 16, 16, 19, 20, 31, 19, 20, 18Mitoses 20, s, 2. 6, 19, 15, 1, 3, 5Area 7, 13, 19, 15, 4, 11, 9, 13, 17, 13Mitoses o, 1, 3, 11, o, 9, 6, 1, 7, 5

Area 7, 9, 6, 10, 12, 11, 10, n , 8, 11Mitoses i , 4, o, 9, 0, o, 7, 4, 3, 1

Averagearea

20

12

95

Averagemitoses

84

4'3

2-9

Mitosesper unit

area

042

0-36

031

The activity of substance X differs from that of proteose solutions in that it isindependent of the presence of plasma, although there is also a marked increase ingrowth in cultures in which plasma forms part of the medium. Proteoses on theother hand are much less active in Tyrode solution. In plasma clots substance Xproduces extensive areas of growth in which the cells are thinly distributed, whereasin proteose solutions of the same nitrogen concentration, e.g. 15 to 30 mg. per100 c.c, the areas of growth are small but compact, and the cells show more fat.

From these results it may be concluded that substance X acts as a stimulant tothe activity of the culture without materially aiding its nutrition. It immediatelyactivates the cells, and lessens the latent period before growth commences. Afterprolonged growth in solutions in which it is the only nitrogenous substance thecells appear starved and shrunken.

Chemically, the active substance is to be classified among the heteroproteoses,or is associated with these bodies. It is insoluble in pure water, but readily solublein salt solutions and in dilute acids and alkalis. It is precipitated by alcohol, butafter such treatment shows reduced activity. It is not heat coagulable, and cansafely be heated to ioo° C. in neutral solution for half an hour. A few experimentswhich have been carried out on the effect of heating solutions of substance X toioo° C. suggest that it is partially inactivated when the fluid is alkaline, but thiseffect may be correlated with the presence of other substances in the solution, sinceit is most marked when the heating takes place prior to the separation of substance Xfrom the bulk of the inactive metaprotein. Table X shows the effect of heatingsolutions of substance X in o-8 per cent. NaCl for half an hour at different hydrogen-ion concentrations. After heating, the solutions were brought back to normal pH,and salt concentrations and their effect tested on growth in hanging drop cultures.

There is evidence that even when kept at o° C. solutions of X slowly deterioratein activity, and freshly prepared solutions give better growth.

Solutions of X have a low surface tension, but its growth-promoting activityis independent of this property. This is shown by the fact that on diluting sufficientlyto render the solution ineffective as a growth stimulant the surface tension remainsat about the same low level.

Utilisation of Proteoses by Chicken Heart Fibroblasts

Table X.

169

Approximate pHof solution

when heated

6 07-08 09 0

Tyrode control

Average growthin two days[Method I]

1 8i-3i-oi-o0 6

130

120

110

100

I 8°"S 70

a 60

§ 50

^ 40

30

20

10

ProteoMpreparation A

Proteosepreparation B

0 10 20 30 40 50 60 70 80Time in hours

Fig. 7. Growth of chick heart fibroblasts in flasks. Solid phase. Plasma 0-5 c.c, embryoextract o-z c.c. and Tyrode solution 0-3 c.c. For details of fluid phase see text.

As already mentioned, all solutions of X which have shown growth-promotingactivity have also given a positive biuret reaction. Although the active substancewas found in the precipitate which forms when strong solutions of Witte's peptoneare dialysed against running water, there was the possibility that the substance wasadsorbed on to the inactive part of this precipitate, and that if dialysed against saltsolutions it might be found capable of passing through a membrane and mightpossibly not give a positive biuret reaction. An experiment was therefore made inwhich solutions of X in o-8 per cent. NaCl were dialysed against o-8 per cent. NaClin collodion sacks. After 24 hours neither the dialysate nor the dialysed solutionshowed any growth-promoting activity, nor any positive biuret reaction, and it was


therefore concluded that the active substance had been completely adsorbed on tothe collodion membrane. These experiments throw no light on the nature orprobable molecular size of the active substance, and it remains undecided whetherit is itself a heteroproteose or is merely adsorbed on to these substances.

Consideration of the methods of preparation used by Baker and Carrel madeit seem probable that their solutions of proteoses would contain the active sub-stance which has been described in the foregoing section. Pure proteoses weretherefore prepared from Witte's peptone in the manner already indicated, and alsoby the method of Baker and Carrel (see Fischer, 1930*) in which the Witte's

130r

120

110

100

9083 80

•g 70

I 60

I 50

< 40

30

20

10

Proteojepreparation A


0 10 20 30 40 50 60 70 80Time in hours

Fig. 8. Growth of chick heart fibroblasts in flasks. Solid phase. Plasma 0-5 c.c,embryo extract o-a c.c. andTyrode solution 0-3 c.c.

peptone was initially dissolved in Ringer's solution and maintained in a highconcentration. The two preparations were tested on cultures in flasks, underexactly similar conditions, the concentration of the solutions being controlled bytheir nitrogen content. The resulting growth (Text-figs. 7 and 8) showed that thepreparation as made by the method of Carrel (solution A) was the more stimu-lating. Steps were therefore taken to separate off from the proteoses prepared inthis way the substances whose solubility was dependent on the presence of salt inthe solution, and which would therefore contain substance X. This result wasachieved by electrodialysis of a proteose solution prepared according to Carrel andBaker after a preliminary dialysis against running tap water and distilled water


until no detectable precipitate was formed on addition of dilute HNOa and AgNO3.The apparatus used was a modified form of that described by Narayanamurti andRamaswami (1930) in which the solution is placed in the annular space betweentwo collodion sacks and distilled water is circulated through the smaller and inner

170

160

150

140

130

120

11000

i 100

1S 80

fi 70

"* 60

50

40

30

20

10

Protcosepreparation A

Solution CProtcose

preparation B

-• Tyrodc

0 10 20 30 40 50 60Time in hours

70 80 90 100

Fig. 9. Growth of io-day chicken heart fibroblasts in flask cultures. The solid phase was madeup as follows. Plasma 0-5 ex., embryo extract 0-2 c.c. and Tyrode 0-3 c.c. Proteose solution A pre-pared after the method described by Carrel (nitrogen — 150 mgm. per 100 c.c). Proteose solution Bprepared after the method described by Carrel followed by electrodialysis and removal of theprecipitate (nitrogen = 150 mg. per 100 c.c). Solution C: extraction of precipitate formed duringelectrodialysis, in o-8 per cent. NaCl solution (nitrogen = 9 mg. per 100 c.c). Tyrode: this curve,actually taken from another experiment, serves to illustrate the degree of activity produced by thevarious solutions.

sack and around the larger and outer sack. The current is passed between a carbonrod cathode in the inner sack and two carbon plate anodes in the water surroundingthe outer. By means of a stirrer driven by a small electric motor the solutionbetween the membranes was kept continuously agitated during the course of thedialysis. In a typical experiment when an E.M.F. of 60 volts was used the current


reached a maximum of 56 milliamps. shortly after the commencement of theprocess, and in 20 hours had fallen to a steady value of 7 milliamps., when thedialysis was stopped. In the proteose solution treated in this way a considerableprecipitate formed. The suspension was centrifuged, and the precipitate extractedwith o-8 per cent. NaCl solution. This extract will be referred to as solution C.A large fraction of the precipitate remained undissolved, and this part was dissolvedin AT/io NaOH, and iV/10 HC1 was added until the first signs of precipitationappeared. The slight precipitate was just removed by NaOH and the calculatedamount of NaCl added to make the concentration of this substance in the solution

130r

Proteosepreparation A


Tyrodesolution

10 20 30 40 50 60Time in hours

70 80

Fig. 10. Growth of io-day chicken heart fibroblasts in flask cultures.Media constituted as in Fig. 9.

o-8 per cent. The pH of this solution was 7-8, and on sterilising a small quantityof material again appeared as a precipitate. This suspension will be referred to assolution D, the electrodialysed proteose solution as B, and the initial proteosesolution prepared according to the method of Carrel and Baker, as A. To solutions Aand B NaCl was added before sterilising to bring the concentration to 0-8 per cent.,and the nitrogen contents of all the solutions were determined. After sterilisationthey were made up as culture media by addition of special Tyrode solution in theway already mentioned.

The effects of the electrodialysed solution were compared with those of theoriginal solution at the same nitrogen concentration and the growth curves obtained


in two different experiments are shown in Text-figs. 9 and 10. Solution B producedgrowth which was both less extensive and also thinner than that produced bysolution A. Similar results were obtained in hanging drop cultures without plasma.They are shown in Table I. It is certain therefore that during the process ofelectrodialysis the solution had lost something of its capacity for promoting growth.Examination of solution C showed that part at least of this loss could be accountedfor by the removal of active substances in the precipitate which had formed. Thesolution C of the first group of preparations, of which the effects of A and B arerepresented in Text-fig. 9, contained only 9 mg. of nitrogen per 100 c.c, yet thecurve, also shown in Text-fig. 9, representing growth in solution C, indicates thatthe areas attained were as great as those in solution B. A very marked differencein the density of the growth was however apparent, for the cells were thinly dis-tributed in the zone of outgrowth in solution C, and much more densely packed insolution B. It seems probable that solution C did not contain adequate nitrogenousnutriment for the cells, but it nevertheless stimulated their activity in a mannerclosely resembling the behaviour of solutions of substance X in hanging dropcultures.

In the case of the second group of preparations, of which solutions A and Bproduced the growth shown in Text-fig. 10, the solutions C and D were tested ata nitrogen concentration of 36 mg. per 100 c.c. The growth of tissues in thesesolutions is expressed graphically in Text-figs. 11 and 12. Text-fig. 12 representsthe growth in the second passage, obtained after subculturing. The outgrowth insolution C was both extensive and well filled with healthy cells, and in the secondpassage its superiority over solution D and over Tyrode solution was clearlyestablished.

It must be pointed out that even the purified proteose preparation B describedabove cannot be considered by any means homogeneous and it is still possible thatits activity may be due to only a fraction of the total material. It was found that onheating the clear electrodialysed solution a marked cloudiness was produced, butthe solution became almost clear again on cooling, and it was not possible to removethe substance responsible for this, since it passed too easily through filter papers,and was not thrown down in the centrifuge. The possibility therefore remains thatthis effect is due to the presence of a small quantity of substance X which mightbe responsible for the slight stimulatory action of proteoses on tissues in saline media.

DISCUSSION.

The future of tissue culture as an experimental method depends very largelyupon whether a more complete knowledge can be obtained of the requirements ofthe cells. The goal at which to aim is a completely synthetic medium whose everyconstituent is under control. This is probably an ideal rather than a possibility,but on the other hand very little information can be obtained upon the nutritionof cells in vitro so long as it is necessary to use such complex systems as plasma andembryo tissue juice in order to obtain extensive and prolonged growth. Not only


110

100

90

s 80

I 70u

"S 60

a 50

E 40Si

** 30

20

10

110

100

90

80

70

60

S 50

e 40

< 30

20

10

ist Passage

Tyrodc

0 10 20 30 40 50 60 70 80 90Time in houre

Fig. i i .

2nd Passage

Tyrode

0 10 20 30 40 50 60Time in hours

70 80 90

Fig. 12.

Figs. 11 and 12. Growth of 10-day chicken heart fibroblasts in flasks. Solid phase. Plasma 0-5 ex.embryo extract 0-2 c.c. and Tyrode 0-3 c.c. C: o-8 per cent. NaCl extract of precipitate formed onelectrodialysis of proteose solution prepared by the method of Carrel. D: Precipitate, after extrac-tion, brought into solution by dilute alkali. Nitrogen concentration 36 mg. per 100 c.c. in botlsolutions.


are these substances complex but they are variable. Plasma, for example, is drawnfrom animals whose metabolism may differ considerably, so that, to mention onlya few possibilities, the hydrogen-ion concentration of different samples may varymaterially, so may the sugar content, the amount of fat present, the protein content,and so on. Thus, although it is possible to keep tissues alive and actively growingfor many years, our knowledge of the factors involved in their nutrition is still inits infancy.

Earlier workers have found that chick heart tissues cannot obtain adequatenutriment for their prolonged growth in vitro either from proteins or amino acids;that is to say, these substances when added to saline media containing glucosedo not allow the cells to live for more than a few days. Amino acids cause increasedactivity of the tissues and appear to be absorbed, but so far, probably owing to thedifficulty of maintaining a constant concentration low enough not to be toxic, theyhave not been found capable of acting as an adequate nitrogen supply. The evidence,however, indicates that the cells are capable of absorbing these substances, whilethey are more or less incapable of utilising proteins.

Carrel and Baker found that growth could be augmented for long periods byadding proteoses to a plasma-containing medium, and suggested that they wereslowly digested, and so supplied the tissues constantly with small quantities ofamino acids. The experiments described in this paper show that this is undoubtedlythe case. The cells themselves are unable to make effective use of proteoses whenthese are given alone in Tyrode solution, either because the proteoses cannotpenetrate into the interior of the cell and the cells do not secrete the necessaryenzymes to break down the proteoses extracellularly in order to absorb them asamino acids, or because even if the proteoses penetrate into the cells they cannot beassimilated. But if plasma is present in the medium, digestion of the proteosesoccurs and they become immediately available to the cells, as is shown by theactivity of the latter. This points to the conclusion that the cells do not secrete inany appreciable quantity enzymes which can attack proteoses, but for this necessarydigestion they depend upon the enzymes in the plasma. It is interesting to note thatproteoses, and not proteins, form the starting-point for this process.

The conclusion with regard to the part played by the plasma in facilitating theutilisation of proteoses by fibroblasts in vitro is supported by the results of theexperiments on growth in hanging drops in the presence of serum. Under theseconditions the proteoses produced a very considerable increase in the number ofmitoses, whereas in media of proteoses and Tyrode solution alone this beneficialeffect was very slight. This suggests that the importance of plasma in enabling thecells to use proteoses effectively is dependent on its chemical nature, and particularlyits proteolytic activity, but not on the mechanical properties of the clot. Serumalone was found to be a much more favourable medium than Tyrode solution,increasing both the areas of growth and the numbers of mitoses to a much greaterextent than a perusal of the literature had led the authors to expect.

Although embryo extract has been found to cause digestion of proteoses, it isnot so active in this respect as plasma, and it is difficult to understand why, in such

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small concentrations, it should exert such a very marked action on the growth oftissues in a medium containing plasma and proteoses. If the digestion by extractfollowed the same course as that brought about by plasma, this alone could notaccount for the greatly increased growth. But there is the possibility, which hasnot yet been investigated, that plasma and extract differ in their manner of digestingthe proteoses and so do not provide the same end-products for the nutrition of thecells. Alternatively the action of the embryo extract may not be primarily digestivebut may stimulate the cell in some other way. The extract perhaps suppliessome other necessary food factor, or possibly a substance which activates themetabolism of the cells so that they migrate and assimilate the protein breakdownproducts more rapidly. There is evidence that such substances do exist, as isindicated by the experiments on substance X described in this paper; this is a bodywhich even in low concentrations greatly increases the activity of the cells, causingthem to migrate out very rapidly from the implant, and to some extent increasingtheir division rate. It may be concluded from the emaciated appearance of cellswhich have grown for any length of time in solutions of substance X, that thissubstance does not provide an adequate nitrogen supply, and moreover it wouldseem unlikely that this particular heteroproteose should supply the cells withnitrogen any more readily than do other proteoses. Other stimulants to cellularactivity have been obtained in the dialysates from embryo extract and egg yolk(Wright, 1926a, 19266). It has been supposed that the former owes its activatingproperties to the presence of amino acids, since its action can in some ways beimitated by mixtures of these bodies. The nature of the dialysate from egg yolkhas not yet been determined, but in hanging drop cultures it produces a very rapidmigration and division rate during the first few days.

Table XI indicates the extent of growth of tissues in various media and offerstentative explanations of their supposed mode of action.

SUMMARY.

1. The growth of chicken heart fibroblasts in physiological saline media, e.g.Tyrode, is not materially increased by the addition of pure proteoses.

2. In the presence of plasma, the addition of proteoses causes increased growthof the tissues, probably owing to the slow digestion of the proteoses to utilisableamino acids by the enzymes of the plasma.

3. If small quantities of embryo extract are also present in the plasma, then thebeneficial effect of the proteoses on the rate of growth is very greatly increased.

4. A thermostable substance has been separated from Witte's peptone, andfrom fibrin digests, which is capable of stimulating cells to increased activity anddivision. The activity of this substance is not dependent on the presence of plasma.The effective agent is associated with, or is actually, a heteroproteose, and its actionis probably not to supply nitrogen, but rather to stimulate the cells in some otherway. It is present in certain preparations of proteoses and partly accounts for theiractivity.


5. Suggestions are put forward to account for the manner in which the variousmedia described affect the rate of growth of the cells.

The authors are deeply indebted to the British Empire Cancer Campaign forgrants which have enabled this work to be carried out. They also wish to recordtheir thanks to Prof. H. S. Raper and to Prof. J. Barcroft, in whose departmentsthey have worked, and to Dr H. B. Fell for facilities at the Strangeways ResearchHospital. Finally they thank Mr Norfield for valuable assistance in supplyingplasma when required.

REFERENCES.

BAKER and CARREL (1925). Journ. Exp. Med. 42, 143.(1926a). Journ. Exp. Med. 44, 387.(19266). Journ. Exp. Med. 44, 397.(1927). Journ. Exp. Med. 45, 305.(1928a). Journ. Exp. Med. 47, 371.(19286). Journ. Exp. Med. 47, 353.(1928c). Journ. Exp. Med. 48, 533.

BURROWS and NEYMANN (1917). Journ. Exp. Med. 25, 93.CARREL (1924). CJi. de la Soc. de Biol. 90, 29.CARREL and BAKER (1926). Journ. Exp. Med. 44, 503.CARREL and EBELING (1921). Journ. Exp. Med. 34, 599.

(1922). Journ. Exp. Med 36, 645.(1923 a). Journ. Exp. Med. 37, 653.(19236). Journ. Exp. Med. 38, 419.(1923c). Journ. Exp. Med. 38, 407.

DEMUTH and VON RIESEN (1928) Biochem. Zeit. 203, 22.EBELING (1924). CJi. de la Soc. de Biol. 90, 31.FISCHER (1930a). Geioebczuchtung, 3 Ausgabe, p. 103.

(19306). Getvebezuchtung, 3 Ausgabe, p. 50.FISCHER and DEMUTH (1927). Arch far Exp. Zellforsch. 5, 131.FISCHER and PARKER (1929). Proc. Soc. Exp Biol. and Med. 26, 585.HAMMETT and REIMANN (1929) Journ. Exp. Med. 50, 445.LAMBERT (1914). Journ. Exp Med. 19, 398.LEWIS (1921). Journ. Exp. Med 33, 485.MAYER (1930). Arch, fur Exp. Zellforsch. 10, 221NARAYANAMURTI and RAMASWAMI (1930). Biochem. Journ. 24, 1650.SPEAR (1931). Brit. Journ. Radiol. 4, 146.STRANGEWAYS (1924). The Technique of Tissue Culture "in vitro" (Heffer), p. 51.VAN SLYKE (1929). Journ. Biol. Chem. 83, 425.WILLMER (1927) Brit. Journ. Exp. Biol. 4, 280.WRIGHT (1926a). Journ. Exp. Med. 43, 591.

(19266). Proc Soc. Exp. Biol. and Med. 23, 603.

JOURNAL OF EXPERIMENTAL BIOLOGY, IX, 2. PLATE I.

Fig. 1. Fig 2

Fig. 3- Fig. 4.

WILLMER AND KENDAL—THE UTILISATION OF PROTEOSES BY CHICKEN HEARTFIBROBLASTS GROWING IN VITRO (pp. 149—79).

JOURNAL OF EXPERIMENTAL BIOLOGY, IX, 2. PLATE II.

%

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Fig. 1. Fig. 2.

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Fig. 4.

WILLMER AND KENDAL—THE UTILISATION OF PROTEOSES BY CHICKEN HEARTFIBROBLASTS GROWING IN VITRO (pp. 149—79).


EXPLANATION OF PLATES.

PLATE I.FIG. 1. Typical growth in serum and Tyrode. io-day-old chick fibroblasts. Hanging drop culture.

(Bouin's fluid and haemalum after 36 hours' growth.)FIG. 2. Typical growth m serum and proteoses. (36 hours' growth )FIGS. 3 and 4. Growth in solutions of substance X. (44 hours' growth )

PLATE II.

FIG. 1. Photograph showing typical appearance of cells growing in solutions of substance X.FIG. 2. Mitosis (telophase) occurring in a cell apparently isolated from all others. The central

implant is below the bottom of the photograph.FIGS. 3 and 4. "Prophase-hke" changes in nuclei of cells in solutions of substance X.

the utilisation of proteoses by chicken …jeb.biologists.org/content/jexbio/9/2/149.full.pdfthe...

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