Cellular interaction and chondrogenesis in vitro

DEVELOPMENTAL.

Cellular

BIOLOGY 21, 584-610

Interaction

(1970)

and Chondrogenesis

BEVERLY BLATT Department

of Biology,

LAVIETES~

Case Western Reserve University, Accepted

in Vitro 1.2

Cleylund,

Ohio 44106

October 6,1969

INTRODUCTION

Analysis of cellular differentiation requires knowledge not only of the biosynthetic capabilities of cells, but also of the limitations placed on these potentialities by the cells’ environment. In culture, the cellular environment consists of nutrient medium and neighboring cells with their secretions. These environmental components may be varied experimentally and the subsequent effect on cultured cells examined. Embryonic chick cartilage cells, for example, show great variability in their response to changes in culture conditions. In some cases when grown at high densities, the cells lose their characteristic cartilage phenotype (Holtzer et al., 1966; Stockdale et al., 1963; Kuroda, 1964a, b; Abbott and Holtzer, 1966a, b; Coon, 1966; Nameroff and Holtzer, 1967; Bryan, 1968a, b). There are, however, other examples where dense cell cultures retain or regain the differentiated appearance of cartilage (Abbott and Holtzer, 1966a; Lavietes and Weston, 1968; Bryan, 1968a, b; Pawelek, 1969; Levenson, 1969). Low density clonal cultures, on the other hand, have been shown to favor phenotypic expression. Yet such cultures will also vary in degree of phenotypic expression depending on the composition of the nutrient medium (Coon, 1966; Abbott and Holtzer, 1968; Holtzer and Abbott, 1968; Pawelek, 1969). It has been suggested that specific cell contact between chondrocytes is required for continued expression of the cartilage phenotype (Abbott and Holtzer, 1966a, b; Holtzer and Abbott, 1966). Cells, plated at the same density, however, in different culture media vary ’ Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology, Case Western Reserve University. * This investigation was carried out under the tenure of a National Science Foundation Graduate Fellowship and supported by USPHS grant 8-ROl-HD-03477 to Dr. James A. Weston. a Present address: Department of Pathology, New York University Medical Center, New York, New York 10016. 584

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in degree of phenotypic expression (Abbott and Holtzer, 1966a; Coon, 1966; Lavietes and Weston, 1968). Clearly then, control of differentiative expression in culture is not determined by a single parameter, but rather by the interaction of several factors. Since these various factors have not been systematically analyzed, this report undertakes to describe the response of populations of embryonic chick sternal chondrocytes to changes in culture medium, cell density, and substrate. MATERIALS

AND

METHODS

Establishment of the cultures. Embryonic chick cartilage cells were obtained from sterna of 13-day White Leghorn embryos using procedures described by Cahn et al. (1967) with the following modifications: (1) The dissociating medium (Roth and Weston, 1967) contained 0.25% collagenase (crude fraction, Clostridium histolyticum, General Biochemicals), 0.25% trypsin (1: 250, Difco), and 10% chicken serum (Gibco) in calcium- and magnesium-free saline. (2) The sterna were never exposed to dissociating medium longer than 40 minutes. (3) Residual clumps remaining after enzyme treatment were removed by filtering the suspension through the stainless steel screen of a Swhmy hypodermic adapter (Milhpore). After appropriate dilution, aliquots of cell suspension were inoculated into a volume of 4 ml of medium in 60-mm Falcon plastic tissue culture dishes. Cultures were maintained at 37’C in an atmosphere of 95% air-5% COZ at saturated humidity. Media were replenished every 2 days. Subcultures were made by the procedures described by Calm et al. (1967). Media. CMRL-1066 (Parker et al., 1957), Ham’s F-10 (Ham, 1963), and Ham’s F-12 (Ham, 1965) as modified by Coon (Cahn et al., 1967) were used. Coon’s modification of F-12 was obtained as liquid ready for use from Gibco. CMRL-1066 and F-10 were obtained from Schwarz BioResearch as dry powder and were reconstituted as needed. After sterilization by pressure filtration through Millipore HA filter, the media were stored at 4°C until use. CMRL-1066 solution was never stored longer than 3 weeks, since older media did not seem to support cell differentiation as well as freshly prepared medium. The appearance of cultures grown in F-10 or F-12 which had been stored 4-5 weeks at 4OC!,on the other hand, did not differ from that of cultures grown in freshly prepared medium. All media were supplemented with 50 units/ml penicillin-strepto-

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mycin (Gibco) and 10% fetal calf serum. After screening several lots of sera, lot No. 070873 (Gibco) was selected and used in all experiments. The serum was stored at -20°C in small aliquots to avoid repeated freezing and thawing. No other complex biological substances, such as embryo extract or bovine serum albumin, were included in the media; and since only one lot of serum was used, differences in phenotypic expression described below must be attributable to differences in the synthetic media. In some cultures, a collagen substrate was used. This was prepared by coating Falcon dishes with reconstituted rat tail collagen, as modified from Bomstem (1958). An acid solution of collagen was added to the dishes, and the excess was removed. The dishes were dried and sterilized by ultraviolet irradiation from a General Electric 30-w germicidal lamp at a distance of 18 inches for 1 hour. Histological preparation. At appropriate times, cultures were rinsed with saline at 37°C and 6xed with glutaraldehyde (6.25% in 0.1 M sodium cacodylate). The fixative was warmed initially to 37’C, but the dishes were subsequently stored at 4°C. After l-24 hours, fixative was replaced with 0.2 M sucrose in 0.1 M sodium cacodylate. The dishes were stored in this buffer at 4OC until stained. After being rinsed with water to remove sucrose, dishes were stained with 0.1% aqueous toluidine blue at pH 7.0 for 2 minutes. They were then rinsed quickly with ethanol (twice with 95% and twice with lOO%), air dried, and covered with a thin layer of immersion oil. Biochemical measurements. Measurements of total protein in the cultures were made by Oyama and Eagle’s modification (1956) of the Lowry method (Lowry et al., 1951). When DNA and protein were to be measured in the same sample, cells were scraped from the dishes with a rubber policeman and extracted by Butter’s method (1967). The extracts were then assayed calorimetrically; DNA by the diphenylamine reaction (Burton, 1956) and protein by the Lowry method (Lowry et al., 1951). Comparison of matrix production. A parameter, the metachromatic index, was devised to estimate the relative proportions of metachromatic and nonmetachromatic regions in a culture. Measurements were made by sampling fixed stained cultures optically, using a modified Chalkley grid (Curtis, 1960) in a microscope ocular. This grid superimposes a circle containing 25 randomly placed dots on the field. The total number of cells within the circle was counted, and

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the number of dots falling on metachromatic or nonmetachromatic cells in each field was recorded. A minimum of 10 fields or 1000 cells was scored for each dish. The fields were randomly selected. The number of hits on metachromatic or nonmetachromatic cells is directly proportional to the area of the culture occupied by each (cf. Curtis, 1960). The ratio of these two parameters defines a “metachromatic index.” A decrease in metachromatic index can occur as the result of either a decrease in metachromatic component or an increase in nonmetachromatic component. The numbers themselves do not allow one to distinguish between these alternatives in analyzing experimental results. However, daily observations and photographic records of the development of the cultures, in conjunction with the numerical data, make such a distinction possible. Statistical comparisons of values obtained were made by testing the null hypothesis that the metachromatic indices of cultures grown under different conditions were equal. An observation of a dot superimposed on a cell is a Bernoulli random variable and follows a binomial distribution. The statistical analysis tests the equality of the proportion of successful Bernoulli trials in independent samples from two populations. Although this sampling method may underestimate the proportion of metachromatic cells where cells surrounded by matrix pile on top of one another, highly significant differences between culture conditions were obtained. RESULTS

Morphology and Growth Media and on Different

of Cartilage

Cultures

in Various Nutrient

Substrates

Differences between cultures established in different culture media but at the same cell density (lo6 cells/66 mm dish) can be seen in the day-by-day development of the cultures (Fig. 1). After 1 day in medium CMRL-1066 supplemented with 10% fetal calf serum, most of the cells appear rounded and refractile by phase microscopy and have not flattened onto the substrate (Fig. la). At this time, on the other hand, cultures from the same suspension at the same density in Ham’s F-10 with 10% fetal calf serum exhibit compact polygonal cells with a few elongated fibroblastic cells interspersed (Fig. le). When fixed and stained with toluidme blue, some metachromasia is associated with most of the cells in both types of culture but only the rounded cells appear encapsulated.

LAVIETES

FIG. 1. The (a-d) cultures F-10 on days contrast optics.

development of cartilage cell cultures in media CMRL-1066 and F-10: in CMRL-1066 on days 1, 3, 5, and 7, respectively; (e-h) cultures in 1, 3, 5, and 7, respectively. Living cultures photographed with phase Scale line = 100 p.

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In cultures grown in CMRL-1066 and fixed on day 3, a fibrous network of metachromatic material appears over clusters of rounded cells, but flattened cells can also be seen under the network peripheral to the metachromatic cells. The fibrous network is seen only in stained preparations. In the living cultures, one can see clusters of refractile cells with peripheral flattened cells which seem to form bridges between refractile clusters (Fig. lb). The cells in the same area 2 days later (day 5) appear multilayered and entirely refractile (Fig. lc). Cultures fixed and stained at this time show reeflike arrays of cells enclosed in metachromatic matrix. The metachromatic material is compact in contrast to the loose fibrous nature of the material in 3-day cultures. In cultures in F-10, epithelia composed of polygonal cells with refractile intercellular material develop by day 3 (Fig If). Elongated fibroblastic cells are also seen scattered throughout the dish. When such cultures are stained, loose fibrous metachromatic material is seen associated with the polygonal cells. By day 5, fibroblastic cells can be seen growing between the polygonal groups, but the refractile polygonal cells do not pile up (Fig. lg). In contrast to CMRL-1066 cultures at this time, the metachromatic material seen in stained F-10 cultures remains loose and fibrous. After day 5, the cultures increase in mass, but no further changes in morphology are observed. The reeflike structures in CMRL-1066 increase in size (Fig. Id, 2a). A few flattened nonmetachromatic cells can usually be seen peripherally. If allowed to continue, further growth results in a confluent metachromatic mat. Confluency is attained by day 12. In F-10 after day 5, both polygonal and fibroblastic cells grow out, and confluency is reached between days 5 and 7. Although some of the polygonal cells may appear very refractile, no extensive multilayering of cells is seen (Fig. lh, 2b). Growth curves, constructed by measuring the total protein content of cultures at daily intervals, show parallel development of cultures in CMRL-1066 and F-10 (Fig. 4). In both media, total protein accumulates rapidly until day 4 and then the rate of accumulation decreases. F-10 cultures, however, do not reach as high values of protein content as CMRL-1066 cultures. This may be correlated with the observation of increased amounts of metachromatic material in CMRL-1066 cultures (Table 1). Since cartilage matrix contains protein, any changes in the rate of matrix production may affect the reliability of a growth curve deter-

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FIG. 2. Cartilage cells after 9 days in culture: (a) in CMRL-1066, (k to different I from the same original suspension respond differently CMRL-l( I66 pile up and are enclosed in refractile matrix. Cells in F-10 confluent sheet; the intercellular spaces are refractile, but the cells a~ Phase car ltrast. Scale line = 100 B.

iIIF ‘-101.c ells edia. c ells in row out int Da not en clos#ed.

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0

0 100

50

: -

0

-

I 2 3 4 5 6 7 8 9 IO DAYS IN CULTURE FIG. 4. Daily accumulation of protein in cultures inoculated with lo6 cells in CMRL-1066 (0) and F-10 (0). Values plotted represent an average of two cultures. The total protein content of each culture was determined and the protein content of the original was subtracted from that value to obtain the value plotted.

mined solely on the basis of protein content. Consequently, daily measurements of DNA and protein in the same samples were also made. In both CMRL-1066 (Fig. 5) and F-10 cultures, DNA and protein curves were parallel and show a log growth phase which levels off between days 3 and 5. For CMRL-1066 cultures, the decrease in growth rate occurs at the same time as the changes in cell morphology (Fig. lb, c) and the texture of the matrix. Cultures were routinely fixed on day 9 and scored for metachromatic index as described. A typical g-day culture grown in CMRL1066 contains large quantities of homogeneous matrix organized around the cells in lacunae, and the cells appear to be piled on top of one another (Fig. 6a). The metachromatic index of such cultures is greater than 3.0. Nine-day F-10 cultures are distinctly different. Cells are arranged as a confluent sheet. Fibroblasts surround clusters of polygonal cells which are associated with diffuse metachromatic material (Fig. 6b). The metachromatic material is not organized FIG. 3. A g-day culture derived from the same original cell suspension seen in Fig. 2 but grown in medium F-10 supplemented with 50 &ml ascorbic acid. A mass of refractile matrix-enclosed cells is seen, but it is surrounded by fibroblastic and p&ygonal cells. The appearance of this culture is intermediate between Figs. 2a and 2b. Phase contrast. Scale same as Fig. 2.

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lO(I-

0 0

0 8

8 W

l

2 2

‘(

)-

0

n 0

0 l 0

0

L ‘0

2

3

4

5

6

7

8

DAYS IN CULTURE FIG. 5. DNA(a) and protein (0) accumulation in CM&1066 cultures. Determinations of both DNA and protein were done on the same sample. The rates of increase seem to remain parallel over the culture period and show a decrease in rate between days 3 and 5. Individual points represent individual cultures.

around lacunae, and the metachromatic index ranges from 0.5 to 1.0. The difference between a metachromatic index of 3.0 and 1.0, with the sample size counted, is highly significant (p < 0.0001). The morphological development of cultures grown in Ham’s F-12, as modified by Coon, differs from that of cultures grown in either CMRL-1066 or F-10. The resultant cultures, when fixed and stained, show metachromasia associated with rounded cells in the center of fibroblastic whorls. This materials, however, is not compactly orga-

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nized. The metachromatic indices were comparable to F-10 cultures (Table 1). Each of these nutrient mixtures is a complex formulation. CMRL1066, however, has many more components than the others. No systematic attempt has been made to identify the components responsible for the differences seen in the behavior of the cells, but one

FIG 6. Cartilage cell cultures fixed with glutaraldehyde after 9 days in culture, stained with toluidine blue: (a) culture grown in CMRL-1066, (b) culture grown in F-10. Note the organization of matrix around cells in lacunae (arrow, a) in CMRL-1066. In F-10, the metachromatic material (arrows, b) is amorphous and not organized in this way. Scale line = 100 p.

TABLE 1 EFFECT OF DIFFERENT MEDIA AND SIJRSTRATRSON METACHROMATIC INDEX OF 9-DAY CULTURIS Average metachromatic Medium

CMRL-1066 F-10 F-12 (Coon’s) a n = number of dishes scored.

index

n”

3 3 3

collaPen

Plastic

3.71 0.70 0.67

3.65 0.98 0.82

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obvious difference in the media seemed likely to affect the deposition of matrix. CMRL-1066 contains 50 Kg/ml ascorbic acid, and the other media have none. When cells were grown in F-10 supplemented with 50 pg/ml ascorbic acid, the metachromatic index scored in fixed g-day cultures, 1.16, was significantly higher (p < 0.0001) than the F-10 control value, 0.77. Piled up clusters of refractile cells were seen among the fibroblasts (Fig. 3). Although ascorbic acid improved the degree of differentiation seen in F-10 cultures, the phenotypic expression observed did not reach the level observed in CMRL-1066 cultures which had a metachromatic index of 3.6. These results suggest that ascorbic acid may play a role in cartilage expression in vitro. Although no attempt has yet been made to grow cells in CMRL-1066 without ascorbate, the observation that CMRL1066 loses its effectiveness when stored as a liquid may correlate.with the known lability of ascorbate in solution. Ascorbic acid, moreover, is required for maximal synthesis of collagen, a major component of cartilage matrix (Robertson, 1961). The presence of collagen, furthermore, has been shown to influence the differentiation of several developmental systems in vitro, e. g., muscle (Konigsberg and Hauschka, 1965) and salivary gland (Kallman and Grobstein, 1964; Grobstein and Cohen, 1965). Thus it was considered possible that some or all of the cells in CMRL-1066 cultures might secrete collagen and that this proposed collagen deposition facilitates further differentiation of cartilage in vitro. If this were so, and if cells in F-10 or F-12 did not synthesize sufficient collagen for lack of ascorbic acid, coating dishes with a collagen film might increase the level of differentiation of cultures in F-10 or F-12. This hypothesis was tested by growing cells in dishes coated with reconstituted rat tail collagen and comparing the response in the three media (Fig. 7, Table 1). Although some changes in morphology were observed with the same medium on different substrates, the corresponding metachromatic indices after 9 days in culture were not increased significantly in any of the media with collagen substrate. These results do not, however, exclude the possibility that collagen and collagen deposition play an important role in matrix production and packaging. Under the above experimental conditions, the collagen was dried onto the dish surface from an acid solution. The arrangement of the collagen molecules would be expected to be random in this case, but cellular response to the presence of collagen may require that collagen be polymerized as ordered fibrils, perhaps

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FIG. 7. Cartilage cells from the same suspension grown in CMRL-1066, F-10, and Coon’s modification of F-12 on plastic and collagen substrates, after 9 days in culture. (a) CMRL-1066, on plastic; (b) F-10, on plastic; (c) F-12, on plastic; (d) CMRL-1066, on collagen; (e) F-10, on collagen; (fj F-12, on collagen. In CMRL-1066 on collagen, reefs are not distinct and the cells tend to form a refractile sheet. In F-10, no differences are seen between cultures on plastic or collagen. In Coon’s F-12 on plastic, isolated fibroblastic whorls with refractile spots are seen; on collagen, a confluent sheet of fibroblastic cells forms, interspersed with nests of refractile cells. Phase contrast. Scale line = 100 p.

in closer association with the matrix-producing cells themselves. This is consistent with current concepts of connective tissue structure as an intimate complex of collagen fibrils and protein polysaccharide chains (see Schubert and Hamerman, 1968, for review).

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Variation of Cell Density Although large variations in cell density have been shown to have profound effects on the degree of cartilage expression observed in vitro (Coon, 1966), the precise relationship between the degree of cellular interaction and phenotypic expression has not been eluci-

FIG. 8. The development of a colony from a single cell in CM%-1066; (a-k) days 2-12 of culture, respectively. The cells spread out before a large refractile maes appears in the center of the colony. The appearance of the clone on day 12 (k) is similar to the reefs in cultures inoculated with 10” celle, seen in Figs. 2a and 7a. Phase contrast. Scale line = 100 p.

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TABLE

Initial

lo6 lo5 lo4 10”

cells cells cells cells

ON METACHROMATIC

Average metachromatic Days in culture

inoculum

597

VZTRO

2

OF CELL DENSITY AND DURATION OF CULTURE INDEX IN CMRL-1066

EFFECT

IN

index”

9

13

17

21

3.25 1.20 3.33 3.25

5.72 0.95 1.74 3.11

3.75 (1) 0.74 0.72 1.68

0.50 0.80 0.81

a AI1 values represent the average of 2 cultures except where noted in parentheses.

dated for cartilage cells in primary cultures. The effects of cell density in cultures in CMRL-1066 were, therefore, examined. Dishes were inoculated with 106, 105, 104, 103, and 10” cells and followed for 21 days. Cultures inoculated with lo6 cells represent the standard mass culture conditions described above. Clonal cultures (10’ cells per dish) produced cartilage-making colonies which stained metachromatically and had lacunae. The plating efficiency was low, never exceeding 2055, a value typical of clonal cartilage cultures without embryo extract in the medium (Coon and Calm, 1966). Metachromatic index was not measured in clonal cultures because such small numbers could not easily be sampled randomly. The growth pattern of these compact colonies seemed to parallel the pattern of growth in cultures inoculated with lo6 cells. Figure 8 shows the life history of a clone from a single cell. The single cell divides several times (Fig. 8a-e), and then these rounded cells flatten and spread (Fig. 8f-h). Subsequently, rounded cells reappear (Fig. 8h-i), and refractile mass piles up in the center of the colony (Fig. 8j-k). Since the extremes of the cell density range produced either masses or colonies of cartilage, it seemed reasonable to expect that intermediate densities would simply give rise to intermediate-size cartilage nodules. This prediction was not confirmed (Table 2). In cultures inoculated with lo5 cells per dish, rounded refractile cells did predominate on the first 3 days of culture, although polygonal and fibroblastic cells were seen. On day 4, however, rounded cells began to loosen, retaining only a tenuous attachment to the dish, while polygonal and fibroblastic cells began to form clusters.

LAVIETES

FIG. 9. dish. ‘IFhe blaetic ccl

day culture in CMRL-1066 from en initial inoculum Oi F ld cella per at the right points to epithelial cells; the arrow at tl le left, tofi broPha se contrast. Scale line = 100 fl.

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On succeeding days, large numbers of loosened cells detached completely from the substrate and floated in the medium. By day 7, large groups of fibroblastic cells had become established and small patches of a different type of cell appeared. The new cells were broad, flat, epithelial, and nonrefractile. After 10 days, no floating cells were seen, but the culture had become a dense melange of rounded refractile, epithelial, and fibroblastic cells. Figure 9 shows a culture on day 12 which had been inoculated with lo5 cells. One can see fibroblastic and epithelial cells surrounding the refractile cells. Fixed stained cultures showed that metachromasia was associated with the rounded cells, and the larger metachromatic clusters had cells in lacunae. The metachromatic index on day 9 was low and decreased further with time (Table 2). The epithelial cells, which were sometimes faintly metachromatic, seemed to arise from the floating cells that had resettled. when floating cells were withdrawn and plated in a fresh dish, a culture developed composed almost entirely of these variant cells (Fig. 10). Many of these cells were multinucleate or multinucleolate. Cultures inoculated at lo4 cells per dish behaved much like cultures inoculated with lo5 cells. The timing and extent of appearance of the various cell types, however was slightly different as a result of the decreased cell number. It took longer for the clusters of fibroblasts to develop, and during this time the rounded refractile cells also grew extensively. This is reflected in the delayed decrease of metachromatic indices (Table 2). At day 9, the value of lo4 cell cultures was comparable to standard lo6 cell cultures but then fell off rapidly as the number of fibroblastic cells and epithelial cells increased. In cultures of 10” cells per dish, individual colonies developed which appeared refractile and matrix producing. Metachromatic indices measured on days 9 and 13 confirmed this. However, as the colonies increased in size, fibroblastic and epithelial cells began to grow at the periphery of colonies, filling the spaces between them.

Rc. 10. Culture inoculated with floating cells from 7-day cultures originally inoculated with lo5 cells in CMRL-1066 6 days after replating. Arrows indicate multinucleate cells. Phase contrast. Scale same as Fig. 9. FIG. 11. A lo-day secondary culture from a IO-day-old primary culture in CMRL1066. The top arrow points out a multinucleate cell; the bottom arrow, a large cell with 4 nucleoli. Phase contrast. Scale same as Fig. 9.

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The metachromatic index declined with the increase in nonmetachromatic component (Table 2). In summary, well-differentiated cartilage appeared in cultures at each density, but differences were seen in the proportion of cells participating in cartilage synthesis. At the extremes of the density range (lo6 and 10’ cells per dish), essentially all the cells were part of cartilage masses. At intermediate densities, however, cartilage nodules were surrounded by nonmetachromatic epithelial and iibroblastic cells. The results of a similar density experiment in F-10 are reported in Table 3. The higher-density cultures maintained the same levels of metachromasia through the duration of the culture period. At intermediate densities, initially high values of metachromatic index fell off with time. This decrease, similar to that found in CMRt-1066 cultures, was correlated with peripheral growth of nonmetachromatic cells. In many reports where cultures of greater than clonal density showed poor differentiation, the cells were inoculated at densities roughly equivalent to lo5 cell&O-mm dish (Abbott and Holtzer, 1966a, b; Nameroff and Holtzer, 1967). The morphology of clonal cultures grown in F-10 also show similarities to morphological features of lo6 cell cultures in the same medium. As in the dense cultures, the metachromatic material is loose and looks like a network entrapping the cells rather than a compact mass enclosing them. Turbulence in the medium caused by feeding or washing the cultures for fixation can sweep away the refractile centers of the colonies (Fig. 12a). Moreover, large colonies in F-10 usually show cellular differentiation within a clone; the central refractile area grades into a peripheral fibroblastic ring (Fig. 12b). The TABLE 3 EFFECT OF CEU DENSITY AND DURATION OF INDEX IN F-10

Initial

lo6 lo5 10’ 10”

’ Average of 2 cultures.

MFFACHROMATIC

Average metachromatic index” Days in culture

inoculum

cells cells cells cells

CULTURE ON

9

13

17

21

0.50 0.44 1.27 4.08

0.96 0.49 0.96 2.05

0.43 0.44 0.66 0.47

0.57 0.49 0.69

FIG. 12. Clones from corresponding cultures in F-10 and CMRL-1066 after 21 days in culture: (a) the central area of a large clone in F-10 where part of the refractile cells have been washed away and are being replaced with fibroblastic cells; (b) an area peripheral to that seen in (a), the refractile polygonal cells grade into a fibroblastic ring; (c) the edge of a clone in CMRL-1066. Clones in F-10 show differentiation of cell types within a colony, refractile centers surrounded by peripheral fibroblastic rings. In CMRL-1066, all but a few flattened peripheral cells are enclosed in matrix. Phase contrast. Scale line = 100 8. 601

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colony in Fig. 12a has lost most of its refractile center, and fibroblastic cells have begun to fill in the empty central area. Figure 12b shows an area of the same colony toward the periphery, where refractile cells merge into the fibroblastic ring. In contrast, Fig. 12c shows the periphery of a colony grown in a corresponding culture in CMRL1066. Except for a few polygonal cells at the periphery, the colony consists of tightly packed refractile cells. Length of Time in Culture The cell density experiments suggested that cells cultured at lo6 cells per dish maintain a constant level of phenotypic expression throughout the period of primary culture. On the other hand, less dense primary cultures showed an increasing accumulation of nonmetachromatic cells with time. Since Coon (1966) had shown that chondrocytes in F-10 retained the ability to express their phenotype over several passages in culture at both mass and clonal density, it was of interest to determine whether phenotypic expression would be maintained through a series of subcultures in CMRL-1066 at high density. Suspensions of cells derived from both CMRL-1066 and F-10 cultures were subcultured every 10 days and tested in both media at each passage. The results are seen in Table 4. Secondary cultures in CMRL-1066 (Fig. 11) were very similar in appearance to primary cultures inoculated with lo5 cells (Fig. 9). This is not surprising since cells which float off and resettle in the same dish at this density may be considered an autonomous subculture by the cell. Epithelial, fibroblastic, and rounded metachromatic cells were all seen. MetaTABLE 4 METACHROMATIC INDEX ON NINTH DAY OF SERIAL MASS CULTURE Metachromatic index Culture Medium”

Passage

1 2 3 4

FF

FC

0.92 0.19 0.02 0.004

0.51 0.28 0.029

cc

CF

3.20 0.65 0.22 0.004

0.14 0.16 0.015

a F = F-10, C = CMRL-1066. The first letter refers to the medium in which cells were cultured in the previous passage; the second letter, the medium of the present passage.

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chromatic material radiated out from distinct lacuna-enclosed cells forming a haze over nearby epithelial cells. Secondary cultures in CMRL-lOf56 of cells originally grown in F-10 had a morphology distinctly different from secondary F-10 cultures in F-10. The cells were broader, and an increased amount of metachromatic material was clearly associated with cells, many of which were enclosed in lacunae. Tertiary cultures showed further decreases in metachromatic index, but again F-10 cells transferred to CMRL-1066 produced values comparable to cells carried in CMRL-1066 all along. In the fourth ~passage, all cultures were predominantly fibroblastic and the metachromasia so rare that the values given in Table 4 have little significance. By the fifth passage, all cultures were entirely fibroblastic. DISCUSSION

The complete cartilage phenotype is most accurately defined as a tissue of cells organized within a matrix. By this definition, the synthesis of chondroitin sulfate, the elaboration of matachromatic material, or the incorporation of sulfate-“% represent only part of the cartilage phenotype-the functions performed by individual cells. Histogenesis, the organization of cells and matrix in relation to each other, on the other hand, requires intercellular cooperation. This study has been concerned with the parameters of culture in vitro that result in histogenesis or tissue reconstruction from a cell suspension. The interaction of factors controlling cartilage expression in culture were shown by varying one factor while holding the others constant, e.g., inoculating equal aliquots of cell suspension into different nutrient media or varying the number of cells inoculated into one type of nutrient medium. The use of the metachromatic index to estimate the relative proportions of matrix present in cultures had some advantages over chemical measurements of matrix components in cell extracts. At the same time estimates of the differences in amount of metachromatic material present were made, differences in the distribution and qualitative appearance of the matrix could also be observed. When differentiated cartilage is defined as a tissue composed of cells and intercellular matrix, the organization of cells and matrix in relation to one another must also be taken into account. Medium Effects Although all primary cultures inoculated with lo6 cells contained rounded cells surrounded by metachromatic extracellular material,

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only in cultures in CMRL-1066 was the matrix organized around lacunae. Since at least some chondrocytes grown in F-10 are able to synthesize chondroitin sulfate (Shulman and Meyer, 1966), CMRL1066 must contain some additional factor(s) involved in matrix packaging. Comparison of the compositions of the media suggested that ascorbic acid, which is present in CMRL-1066 but not F-10 or F-12, might be such a factor. When ascorbic acid was added to F-10, matrix accumulation and organization were significantly increased. Recently, Levenson (1969) obtained a similar result with the same concentral tion of ascorbic acid added to cultures of chick limb cartilage and Meckel’s rod cartilage in Eagle’s medium. Levenson (1969) also suggests that collagen may be related to the improved differentiation obtained in the presence of ascorbic acid (cf. first section under Results). While ascorbic acid is necessary for extensive collagen synthesis (Robertson, 1961), it is doubtful that the vitamin has any stimulatory effect on chondroitin sulfate synthesis (Koefoed and Robertson, 1966). A comparison of the elaboration of extracellular matrix in CMRL-1066 and F-10 cultures by electron microscopy together with an analysis of the rates of collagen and chondroitin sulfate synthesis under corresponding conditions should provide more information about the role of collagen in matrix formation. Cellular Interaction

in Primary

Cultures

Previous workers have shown that clonal cultures of embryonic chick chondrocytes are phenotypically expressive but that monolayer cultures at higher densities appear to dedifferentiate (Coon, 1966; Abbott and Holtzer, 1966b; Bryan, 1968a, b). The results presented in Tables 2 and 3 largely substantiate these observations. Only CMRL-1066 cultures inoculated with lo6 cells do not conform. This apparent contradiction may be resolved by considering another set of observations. Earlier work has also shown that increased cell density does not necessarily prevent matrix production. When pellets derived from suspension of chondrocytes are grown as organ cultures, the cells continue to make matrix (Holtzer et al., 1960; Stockdale et al., 1963; Abbott and Holtzer, 1966a; Nameroff and Holtzer, 1967). If specific cell contact is necessary for the retention of the cartilage phenotype in pellets, as suggested by Abbott and Holtzer (1966a), then an inoculum of lo6 cells per dish may represent a threshold value such that sufficient cell contact occurs in these cell cultures for maintenance of phenotypic expression.

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One can test this idea by calculating the number of cell contacts that would be expected to occur at the time of plating. The calculation is based on the assumption that the cells are spherical and make one-point tangential contacts with one another and on the known values of cell diameter, dish diameter, and number of cells inoculated. The values generated from such calculations show that with an inoculum of lo6 cells per dish, each cell would be expected to make on the average of 2.5 contacts; at lo5 cells per dish, only 1 cell in 4 would be expected to contact another; at lo* cells per dish, 1 in 40, etc. These numbers suggest a causal relationship between size of cell inoculum and the phenotypic expression observed in the cultures. When lo6 or more cells are inoculated into a 60-mm dish, a situation exists that allows continued cellular interaction from the beginning of the culture period. Thus the differentiative phenomena observed in such cultures are more closely related to pellet cultures than to lower density cell cultures. That cell contact alone is insufficient to guarantee phenotypic expression is seen by comparing cultures in F-10, F-12, and CMRL1066, all inoculated with lo6 cells (Table 1). Yet a nutrient medium that is able to support phenotypic expression can do so only under appropriate conditions of cell contact (Table 2). It is not clear why clonal cultures in which the probability of cell contact at the time of plating is vanishingly small show better phenotypic expression than slightly more dense cultures. This observation may result from the experimental design. Table 2 shows that the length of time required for decreases in metachromatic index to appear increases as cell inocula become smaller. The same processes might also be observed in cultures inoculated with 10” cells if these were maintained longer than 21 days. This expectation would be consistent with data from cultures grown in F-10 seen in Table 3 and Fig. 12 and observations from other laboratories (Abbott and Holtzer, 1968; Holtzer and Abbott, 1968). Tissue Differentiation

and Inherent

Obsolescence

Excessively flattened, multinucleate or multinucleolate cells like those seen in Figs. 9, 10, and 11 have been described by others. Coon (1966) called them “giant” cells; Bryan (1968a, b), “variant” cells; and Abbott and Holtzer (1968), “altered chondrocytes.” Such cells usually appear in long-term cell cultures and have been considered senescent or moribund (cf. Hayflick and Moorhead, 1961). In the

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present study, however, these cells are first seen in subcultured populations (where resettling of floating cells is also considered to be an autonomous subculture) and after day 15 as outgrowth around colonies in cultures inoculated with lo3 cells. These cells which appear so early in the culture period presumably are also senescent, but their senescence seems to be more characteristic of chondrocytes in particular than of cultured cells in general. The cells used in these experiments were derived from 13day sterna. Later in development, the sternum becomes ossified. Perichondrial tissue gives rise to osteoblasts which invade the cartilage matrix, and this invasion results in the gradual erosion of cartilage. Chondrocytes released from their capsules at this time in uiuo quickly disintegrate (Fell, 1925). Cooper and Lash (1964) studied the response of cartilage cells from embryonic chick limbs at different stages of differentiation to monolayer culture and to reaggregation from suspensions and subsequent organ culture. Hypertrophied cells from the diaphysis did not divide, and the cultures died off after 7-8 days. Articular and epiphyseal cartilage, however, continued to proliferate. Aggregates, on the other hand, derived from any of the 3 different suspensions all reconstituted cartilaginous tissue. The 13-day sternum contains these same stages of differentiation: hypertrophied chondrocytes, cells destined to hypertrophy, and cells that remain cartilage in the adult. The cell suspensions used in these experiments presumably contained, therefore, a distribution of cells in various stages of differentiation. The hypertrophied cells in cultures inoculated with lo6 cells might participate in tissue recoustruction as they would in a pellet (Cooper and Lash, 1964). At lower densities, however, those cells which appear senescent might represent hypertrophied cells which were not incorporated into a cartilagesynthesizing group. This includes the resettled floating cells and peripheral epithelial cells. When phenotypically expressive cultures in CMRL-1066 are subcultured, the cells do not reconstitute cartilaginous tissue. This finding may be correlated with Fell’s (1925) observations which suggest that hypertrophied chondrocytes can not survive release from their capsules of matrix. Accordingly, the enzymatic digestion of matrix which is part of the procedure for the preparation of cell suspensions for subculture might be expected to have a deleterious effect on encapsulated cells, A similar loss of phenotypic expression was ob-

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served in pellet cultures which were dissociated and repelleted every 7 days (Stockdale et al., 1963). If cartilage cells in culture undergo inherent obsolescence, how were Coon (1966) and Bryan (1966a, b) able to maintain chondrogenic strains over 30 cell generations? Perhaps this was possible because in their cultures grown in F-10, the cells secrete metachromatic material but do not form capsules (see Bryan, 1968a, Fig. 2). It is the formation of the capsule, however, that seems to establish the pattern of senescence(Fell, 1925; Cooper and Lash, 1964). Pawelek (1969) has shown that cartilage differentiation in vitro is stimulated by low oxygen tension in the presence of thyroxine. He suggests that these factors may control the pattern of cartilage differentiation in uiuo. If this be so, the cultures maintained in CMPL1066 under low oxygen tension might not undergo the degradative changes characteristic of ossifying cartilage in uiuo. Preliminary experiments seem to confirm this prediction (Lavietes, unpublished observations). The Cartilage Phenotype

in Vitro

High-density cultures of embryonic chick cartilage cells in CMFtL1066 possess several properties which enhance their usefulness for the study of embryonic differentiation. First, the cultures undergo tissue reconstruction in the absence of embryo extract or hormone supplement (cf. Levenson, 1969; Pawelek, 1969). Second, essentially all the cells in the population participate in the expression of phenotype. Third, the cells can function as chondrocytes for only a limited time in culture before they proceed to senescence without adapting themselves to in vitro conditions. The cells in vitro seem, therefore, to parallel closely the behavior of cells in uiuo. Hence, this system may be of value in providing a means to study the transition between two fundamental levels of organization-the cell and tissue. SUMMARY

The elaboration of organized tissue structures by dissociated chondrocytes has been analyzed in uitro. Such tissue reconstruction involves not only synthesis of matrix components, but also the organization of matrix around the cells in lacunae. The expression of this tissue phenotype was favored when an initial inoculum of at least lo6 cells per dish was grown in nutrient medium CMRL-1066 supplemented with 10% fetal calf serum.

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At the same density in Ham’s F-10 or F-12 (as modified by Coon) with the same serum supplement, the cells produced less matrix; and the matrix produced was not organized around the cells. Growth on a collagen substrate did not alter the cells’ response to the various media. However, the degree of differentiation in F-10 was enhanced by supplementing the medium with ascorbic acid. In clonal cultures (10’ cells per dish) in CMRL-1066, well-differentiated colonies arose. The development of these colonies paralleled cultures inoculated with lo6 cells. Cultures established at intermediate cell population densities in the same medium, however, developed large areas of cells not associated with organized metachromatic matrix. It is suggested that in CMRL-1066 cultures, chondrocytes not only proliferate, secrete, and organize matrix, but also degenerate like hypertrophied cartilage cells in uivo. Because the cells interact as a population but do not adapt to in vitro conditions, this culture system may be useful for studying the processes involved in the transition from the cellular to the tissue level of organization. Thanks are due Dr. Douglas Davidson and Dr. Helen H. Evans for their help with this investigation and the manuscript. I am especially grateful to Dr. James A. Weston for providing me with a constant supply of invaluable counsel and pertinent criticism. REFERENCES J., and HOLTZER, H. (1966a). The loss of phenotypic traits by differentiated cells. III. Reversible behavior of chondrocytes in primary culture. J. Cell Biol. 28,

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413-487. J., and HOLTZER, H. (1966b). Differences in phenotypic expression of chondrocytes grown in monolayers or in clones. Am. Zoologist 6, 548. ABBIXT, J., and HOLTZEFI, H. (1966). The loss of phenotypic traits by differentiated cells. V. The effect of 5-bromodeoxyuridine on cloned chondrocytes. Proc. N&l. Acad. Sci. U.S. 59, 1144-1151. BORNSTEIN, M. B. (1956). Reconstituted rat tail collagen used as a substrate for tissue culture in Maximow slides or roller tubes. Lab. Inuest. 7, 134-140. BRYAN, J. (1966a). Studies on clonal cartilage strains. I. Effect of contaminant noncartilage cells. Exptl. Cell Res. 52.319-325. BRYAN, J. (1968b). Studies on clonal cartilage strains. II. Selective effects of different growth conditions. Zhptl. Cell Res. 52, 327-337. BURTON, K. (1956). A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Rio&em. J. 62, 315-323. CAHN, R. D., COON, H. G., and CAHN, M. B. (1967). Cell culture and cloning techniques. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 493-530. Crowell, New York.

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