J. Cell Sci. 41, IS9-I75 (1980) 159Printed in Great Britain © Company of Biologists Limited ig8o

CELL PROLIFERATION AND MIGRATION ON

COLLAGEN SUBSTRATA IN VITRO

SETH L. SCHOR

Cancer Research Campaign Department of Medical Oncology, Christie Hospital andRadium Institute, Wimslow Rd, Manchester M20 gBX, England

SUMMARY

Quantitative data are presented regarding cell proliferation and migration on (a) collagenfilms (b) the surface of 3-dimensional gels of native collagen fibres and (c) within the 3-dimen-sional collagen gel matrix, as part of a study of the effects of the extracellular matrix on cellbehaviour. The nature of the collagen environment was found to influence the proliferation ofcertain cell types, but not of others. For example, HeLa cells proliferate at approximately thesame rate and reach the same saturation cell densities on all of the collagen substrata, whilehuman skin fibroblasts grow more slowly within the 3-dimensional collagen gel matrix com-pared with cells either on the gel surface or on collagen films. The 3-dimensional gels of nativecollagen fibres may also be used to study cell migration on the gel surface, as well as cellmigration (or 'infiltration') from the gel surface into the 3-dimensional collagen matrix. Twomethods have been used to obtain quantitative information concerning cell infiltration intothe collagen gel, one involving the selective removal of cells from the gel surface, while theother relies on direct microscopic examination. Of the cells examined to date, epithelial cells(both normal and tumour) do not show infiltrative behaviour, while both normal and virallytransformed fibroblasts, as well as tumour cells of non-epithelial origin (e.g. melanoma), doinfiltrate into the collagen gel matrix, at rates which vary considerably according to cell type.

INTRODUCTION

In recent years it has become apparent that the extracellular matrix does not func-tion merely as an inert structural support, but plays an important role in the controland integration of cell behaviour in multicellular organisms (Grobstein, 1975).This has been best documented in terms of the effects of the extracellular matrix oncell differentiation. Collagen is a major constituent of the extracellular matrix invivo and collagen substrata in vitro have been shown to influence the differentiationof a number of cell types, including myoblasts (Konigsberg & Hauschka, 1965),chondrocytes (Kosher & Church, 1975), liver parenchymal cells (Michalopoulos& Pitot, 1975), mammary epithelial cells (Emerman & Pitelka, 1977), corneal epithelialcells (Meier & Hay, 1975), Schwann cells (Bunge & Bunge, 1978), and guinea-pigepidermal cells (Murray et al. 1979). In each of the above studies, the effects of thecollagen substrata were monitored either by noting changes in cell morphology or,more usually, by measuring the synthesis of macromolecules characteristic of thedifferentiated phenotype.

In addition to its influence on cell differentiation, the extracellular matrix may alsoplay an important role in the control of cell proliferation and migration (Grobstein,1975; Noden, 1975). Although studies in vitro have suggested that collagen may affect

160 S. L. Schor

cell proliferation (Ehrmann & Gey, 1956; Gey, Svotelis, Foard & Bang, 1974;Liotta et al. 1978) and migration (Stenn, Madri & Roll, 1979), quantitative data havenot been presented. The main purpose of this communication is to describe techniqueswhereby such quantitative data may be simply obtained and to present informationconcerning the proliferation and migration of various cell types on collagen.

Several different types of collagen have been identified in vivo (Grant & Jackson,1976; Miller, 1977) and collagen extracted from various tissues has been used toform a number of different substrata in vitro (Ehrmann & Gey, 1956; Klebe, 1975;Rubin, Oldberg, Hook & Obrink, 1978; Elsdale & Bard, 1972). In the present studytype I collagen extracted from rat tail tendons has been used to form collagen filmsand 3-dimensional gels of native collagen fibres as previously described (Schor &Court, 1979).

MATERIALS AND METHODS

Cell cultures

Stock cultures of HeLa cells (obtained from Dr R. T. Johnson, Zoology Department, Uni-versity of Cambridge) BKH21/C13 (obtained from Imperial Cancer Research Fund, LincolnInns Fields, London), human adult skin fibroblasts, passages 4 to io, (established in thisLaboratory) and RPMI-3460 Syrian hamster melanoma cells (obtained from Dr M. Steinbeig,New York University Medical Center) were grown in plastic dishes in Eagle's MEM growthmedium supplemented with either 10 or 15 % foetal calf serum, 2 mM glutamine, 1 mMsodium pyruvate, non-essential amino acids (Gibco-biocult) and 100 units/ml of penicillin andstreptomycin. Stock cultures were subcultured once a week and the medium changed 3 times aweek.

Preparation of collagen substrata and cell cultures

Rat tail tendons (70 g) were extracted with 05 N acetic acid for 2 days at 4 °C. The collagensolution in acetic acid was then centrifuged at 2000 g for 3 h to remove undissolved debris andthe supernatant mixed with an equal volume of 20 % NaCl to precipitate the collagen. Thecollagen precipitate was recovered by another centrifugation at 2000 g for 3 h, redissolved in500 ml of 0-5 N acetic acid and then dialysed against 10 x volume of distilled water for 2 days,changing the distilled water twice per day. Finally, this aqueous solution of collagen wascentrifuged overnight at 2000 g and the clear supernatant stored at 4 °C.

The optical density of the aqueous collagen solution was measured at 230 nm and the con-centration of collagen calculated using a calibration curve prepared from standard solutions offreeze-dried rat tail tendon ccllagen extracted in the same manner. The concentration of colla-gen in the aqueous stock solutions was adjusted to between 20—2-5 mg/ml prior to use.

Three-dimensional gels of native collagen fibres were prepared in 35-mm plastic tissueculture Petri dishes (Gibco biocult, Ltd, Uxbridge, cat. no. 53066) by rapidly mixing 8-5 mlof the collagen solution with 1 ml of 10 x concentrate MEM and 0-5 ml of 4'4% sodiumbicarbonate and pipetting 2-ml aliquots into the dishes. Films of collagen were prepared bycovering the surface of 35-mm plastic tissue culture Petri dishes with the solution of collagen,removing excess liquid with a Pasteur pipette and incubating the dishes for 20 min in a desic-cator containing an open beaker of concentrated ammonia. The films were then air-dried andwashed 5 times with distilled water before use. The arrangement of collagen molecules withinthese films has been discussed in Schor & Court (1979).

In addition to growing cells on the surface of the collagen gels, it is possible to incorporate asingle cell suspension within the 3-dimensional gel matrix when the gel is formed simply byadding a cell suspension to the collagen-growth medium mixture before plating it into thedishes. It should be noted that cells so incorporated within the gel are actually attached tocollagen fibres and are not in suspension, as are cells in soft agar.

Cell proliferation and migration on collagen 161

Determination of total cell number

Cells growing on plastic dishes or films of collagen were first washed 3 times with Hanks'balanced salt solution (Gibco-biocult, Ltd, Uxbridge) and then incubated for 10 min at 37 °Cwith 2 ml of 0-05 % trypsin plus 2 mM EGTA (ethyleneglycol-bis(/?-aminoethylether) N,N-tetraacetic acid) in Dulbecco's phosphate-buffered saline (PBS). The trypsin solution wasthen pipetted up and down several times to detach all cells from the dishes, the resultant singlecell suspension placed in a vial with 5 ml of 'Isoton' (Coulter Electronics, Ltd., Harpenden,Herts), and the dishes washed with 3 ml of Hanks' balanced salt solution, which was thenadded to the vials to give a final volume of 10 ml. The number of cells/ml was determined usinga Coulter electronic particle counter.

Cells growing on and within the 3-dimensional collagen gels were washed 3 times with Hanks'balanced salt solution and then incubated with 2 ml of 0-2 mg/ml collagenase (Sigma Ltd.,cat. no. C2139) in serum-free MEM for 5 h to dissolve the gels completely. The dissolved gel(with any floating cells) was then centrifuged at 300 g for 10 min and the small cell pelletresuspended in 2 ml of 005 % trypsin plus 2 mM EGTA in PBS, which was then added backto the appropriate dishes to detach those cells which had adhered to the plastic as the collagengel was dissolved. Dishes were incubated for 10 min at 37 CC and cell number then determinedas described above for cells growing on plastic dishes or collagen films.

Determination of trypsin sensitivity and the total number of cells growing on the surfaceof the ^-dimensional collagen gels

Cell cultures growing on the surface of the 3-dimensional collagen gels were washed 3 timeswith the Hanks' balanced salt solution and then incubated with 2 ml of 005 % trypsin plus 2mM EGTA in PBS for 20 min. The trypsin solution was then pipetted up and down severaltimes to detach as many cells as possible. The resultant cell suspension was then transferredto a vial containing 5 ml of Isoton, the dishes washed with 3 ml of Hanks' balanced salt solutionand this added to the vial for determination of cell number with a Coulter electronic particlecounter. The trypsin-treated gels were incubated with 2 ml of 0-2 mg/ml collagenase in serum-free MEM for 20 min at 37 °C. The collagenase solution was then pipetted up and down todetach the remainder of the cells quantitatively from the gel surface while leaving the bulk ofthe gel intact. The cell suspension in collagenase was added to a vial containing 5 ml of Isoton,the gels washed with 3 ml of Hanks' balanced salt solution, which was added to the appropriatevials for counting. The total number of cells on the gel surface is the sum of the cells detachedby trypsin and collagenase.

Determination of the per cent of cells within the collagen gel matrix

Coulter counter method. Cell cultures on the 3-dimensional collagen gels were treated asdescribed in the preceding section to remove all cells growing on the gel surface and then wereincubated a second time with 2 ml of 02 mg/ml collagenase in serum-free MEM for 4-5 hto dissolve the gel completely. The number of cells recovered in this fashion (i.e. those cellswithin the gel matrix) were determined as described above for total cell counts on and withinthe collagen gels. The total number of cells per gel culture is determined by adding the numberof cells on the gel surface and the number of cells within the gel matrix and these data used tocalculate the percentage of the total cell number within the gel.

Microscopic method. Unfixed cell cultures on collagen gels were examined using phase-con-trast optics with a Leitz Diavert microscope fitted with a SY2 photographic graticule definingan area of 09 x 065 cm. The number of cells on and below the gel surface within the areadefined by the graticule was determined at approximately 20 areas of the gel surface selectedat random moving in a straight line across the diameter of the dish. These data were then usedto calculate the mean ± s.D. of the total cell number per field and the per cent of cells within thegel matrix. The total cells per field could be converted into absolute terms of total cells per gelculture by calculating the true area of the gel surface appearing in the graticule field at theparticular magnification of the objective lens (e.g. 5 85 x io"3 cm1 for the 10 x objective) and thetotal surface area of the gel (e.g. 96 cm1).

162 S. L. Schor

These two methods for assessing cell infiltration into the gel matrix have given comparableresults with all the cells examined to date, including those which do not appear by microscopicexamination to have moved into the gel. When the Coulter counter was used to determine cellnumber, the s.D.s were routinely less than 12 % of the mean and are therefore not presentedwith the data.

RESULTS

Cell proliferation

The growth characteristics of HeLa cells on plastic dishes, on collagen films, onthe surface of 3-dimensional collagen gels, and within the 3-dimensional gel matrixare shown in Fig. 1. The proliferation rates and saturation densities of HeLa cellswere the same on all of the substrata. The results obtained with BHK cells and early

100

10

6 8Days

10 12 14

Fig. 1. The growth of HeLa cells on plastic dishes and collagen substrata. HeLa cellsin MEM containing 10 % foetal calf serum were plated at an initial density of io4 cells/dish and cell number determined at various times thereafter as described in Materialsand methods. Each point in the figure is the mean of triplicate determinations and theS.D. was always less than 12% (not shown). # , cells on plastic dishes; O, cells oncollagen films; A, cells on the surface of collagen gels; x , cells within the 3-dimen-sional gel matrix.

Cell proliferation and migration on collagen 163

passage human diploid skin fibroblasts are shown in Figs. 2 and 3, respectively.BHK cells grew at a somewhat slower rate on and within the 3-dimensional collagengels compared with collagen films and plastic dishes, although the same saturationcell densities were reached on all substrata. The growth characteristics of adulthuman skin fibroblasts showed more pronounced differences on the various substrata.Growth on the plastic dishes, collagen films and on the surface of the 3-dimensional gels

100

1 1 1 1 1 1 1 1 1 1 16

Days10 12

Fig. 2. The growth of BHK cells on plastic dishes and collagen substrata. BHK cells inMEM containing 10 % foetal calf serum were plated at an initial density of io4 cells/dish and cell number determined at various times thereafter as described in Materialsand methods. Each point in the figure is the mean of triplicate determinations and theS.D. was always less than 12% (not shown). 0 , cells on plastic dishes; O, cells oncollagen gels; A> cells on the surface of collagen gels; x , cells within the 3-dimensionalgel matrix.

occurred at approximately the same rate, while fibroblasts cultured within the 3-dimensional gel matrix showed a significant lag period (2 days) before growth began.The saturation cell densities were again the same on all substrata. Previous observa-tions have suggested that human skin fibroblasts cannot proliferate within the matrixof such 3-dimensional collagen gels (Bard & Elsdale, 1971), although this conclusionwas not based on direct determination of cell number as shown in Fig. 3. The data

164 S. L. Schor

10

"8

8h

6 8Days

10 12 14

Fig. 3. The growth of human skin fibroblasts on plastic dishes and collagen substrata.Human skin fibroblasts (adult, passage 5) in MEM containing 15 % foetal calf serumwere plated at an initial density of 25 x io4 cells/dish and cell number determinedat various times thereafter as described in Materials and methods. Each point in thefigure is the mean of triplicate determinations and the S.D. was always less than 12 %(not shown). # , cells on plastic dishes; O, cells on collagen films; A, cells on thesurface of collagen gel; x , cells within the 3-dimensional gel matrix.

presented in Figs. 1-3 were obtained using concentrations of foetal calf serum sufficientto achieve optimal cell growth on plastic dishes.

Cell migration on the surface of collagen films and gels

Information about the ability of cells to migrate on the different substrata may beobtained from the relative position of cells within individual colonies after severaldays of growth. The data obtained with HeLa cells are shown in Fig. 4; cells wereplated at an initial density of io3 cell/dish and colonies examined 10 days later. Colo-nies of HeLa cells growing on plastic tissue culture dishes consist of tightly packedpolygonal cells, suggesting that daughter cells have not moved appreciable distancesrelative to each other. In contrast, HeLa cells growing on the surface of collagen filmsor 3-dimensional gels of native collagen fibres are separated from each other by dis-tances on average greater than several cell diameters, indicating that cells have migra-ted on the collagen surfaces. Cells initially plated as a single-cell suspension withinthe 3-dimensional gel matrix give rise to rather compact colonies in which the cells liein close proximity to each other. Similar results for HeLa cells growing within the 3-dimensional gel matrix have been reported by Elsdale & Bard (1972). These authorsalso note that although colonies of SV3T3 cells on plastic tissue culture dishes consist

Cell proliferation and migration on collagen

<~7T •

4 A B

**#%

• « - ^

Fig. 4. The appearance of HeLa cell colonies growing on collagen substrata. HeLacells in MEM containing 10 % foetal calf serum were plated on the various collagensubstrata at an initial density of io3 cells/dish and photographed 10 days later. Certaincultures were fixed with 10 % formahn-saline and stained with crystal violet. Typicalcolonies are shown. A, fixed and stained colony on plastic dish; B, fixed and stainedcolony on collagen film; c, fixed and stained colony on collagen gel; D, fixed and stainedcolony with collagen gel matrix; E, living dells on plastic dish (phase optics); F, livingcells on collagen gel (phase optics). Bar, 50 /an.

i66 S. L. Schor

DFig. 5. Human skin fibroblasts growing on and within 3-dimensional gels of native col-lagen fibres. Human skin fibroblasts (adult, passage 4) in MEM containing 15%foetal calf serum were plated on the surface of 3-dimensional collagen gels at aninitial density of 2 5 x io4 cells/dish and photographed 10 days later. Certain cultureswere fixed with 10% formalin-saline and stained with crystal violet, A, fixed andstained cells on the gel surface; B, fixed and stained cells which had infiltrated intothe gel matrix; c, living cells on the gel surface (phase optics); D, the same area of thegel surface as in c after treatment with trypsin and collagenase to remove all cells fromthe gel surface. Bar, 50 fim.

of separate, elongated cells, colonies on the surface of 3-dimensional collagen gels consistof tightly packed cell aggregates (an observation confirmed in our laboratory and incontrast to the behaviour of HeLa cells reported here). These results indicate that themigratory behaviour of cells on collagen substrata may be quite different from that onplastic.

Cell proliferation and migration on collagen 167

Cell migration from the surface of the collagen gel into the 3-dimensional gel matrix

The 3-dimensional collagen gels may also be used to demonstrate the ability ofcells to migrate from the gel surface into the 3-dimensional matrix. For example,cultures of HeLa cells growing on the surface of the collagen gels (as shown in Fig. 4)eventually form a layer of cells covering the entire surface, but no cells migrate intothe 3-dimensional gel matrix. This inability of HeLa cells to move into the gel matrixoccurs in spite of the increased mobility of these cells on the gel surface compared toplastic and their ability to proliferate within the collagen gel when placed there as asingle-cell suspension when the gel is formed (Fig. 1).

Quite different results are obtained with both BHK cells and human skin fibro-blasts. In this case, cells growing on the surface of the collagen gels soon move intothe 3-dimensional gel matrix in addition to forming a monolayer of cells on the gelsurface (Fig. 5). In order to distinguish this behaviour from migration restricted to thegel surface, the movement of cells from the surface into the 3-dimensional gel matrixwill be referred to as 'infiltration'.

A number of cell types have been examined in this fashion and the results arepresented in Table 1. Of the cells examined to date, those which do infiltrate intothe gel matrix include fibroblasts (normal and virally transformed) and tumour cellsof non-epithelial origin. Cells which do not infiltrate into the gel include all epithelialand endothelial cells examined, both normal and tumour.

Quantitative data concerning the infiltration of cells into the three-dimensional gelmatrix

Although data similar to those presented in Table 1 can be used to compare thebehaviour of different cell types in vitro, more precise information regarding the pro-portion of cells within the collagen matrix as a function of time in culture would be ofgreater utility. This type of quantitative information may be obtained by 2 quitesimple, independent methods, one involving the selective removal of cells growing onthe gel surface and the other direct microscopic examination of the intact gel.

Cells may be selectively removed from the gel surface (leaving those cells whichhave infiltrated to the gel matrix undisturbed) by the sequential application of trypsinand collagenase. Cultures are first treated with trypsin for 20 min which removes aproportion of cells from the gel surface (Materials and methods). The proportion ofcells on the gel surface removed by treatment with trypsin is not constant, but variesas a function of cell type and time in culture (Schor & Court, 1979). The cells re-maining on the surface of these trypsin-treated gels are then completely removed byexposure to collagenase for 20 min, leaving the bulk of the gel intact. Fig. 5 c, Dshow the appearance of human skin fibroblasts growing on the surface of a 3-dimen-sional collagen gel and the same area of the gel after treatment with trypsin and colla-genase to remove all the cells from the surface. The fact that this treatment completelyremoves all the cells from the gel surface is easily ascertained by microscopic exami-nation (phase optics) and has been confirmed with the scanning electron microscope(Dr T. Allen, personal communication). All of the cell types which have been

168 S. L. Schor

Table i. The ability of various cell types to infiltrate into the 3-dimensionalgel matrix

Cell infiltration into the collagen gel matrixK

No Yes Degree

Cell lines Cell linesHeLa (cervical carcinoma) BHK (baby hamster kidney fibroblasts) +Hep II (larynx carcinoma) PyBHK (polyoma virus-transformed BHK) +Chang liver (human liver) 3T3 (mouse embryo) +

Early passage SV3T3 (SV40 virus-transformed 3T3) +Chick embryo sternal chondrocytes CHO (Chinese hamster ovary) +Rat liver parenchymal cells RPMI 3460 (Syrian hamster melanoma) + + +Rat hepatoma Early passageEndothelial cells Human skin fibroblasts (adult) + +

(a) Pig aorta Human skin fibroblasts (foetal) + + +(b) Cow brain Human melanoma ±(c) Human umbilical vein Human glioma + +

Human normal kidney cellsHuman renal carcinoma Human fibrosarcoma + +

(hypernephroma)Human mammary carcinoma Mouse peritoneal macrophages (resident) + +Human bronchial carcinomaNormal human epidermal cells

Cells were plated onto the surface of 3 -dimensional collagen gels and maintained in culturefor 10-14 days. Cell lines were in medium containing 10% foetal calf aerum and all earlypassage cells in medium containing 15 % serum. Of the cells examined to date, certain onesgrow to form a monolayer or multilayer on the gel surface, but do not infiltrate into the 3-dimensional gel matrix ('No' column). Other cells do infiltrate into the gel matrix ('Yes'column) at different rates. The percentage of the total cell number within the gel matrix after10-14 days of growth can be expressed in approximate terms ('degree' column), so that+ = 1-10%, 4- + = 20-30% and + + + = > 30%. The data shown for the humantumours are based on few samples (up to 5 per tumour type) and more detailed informationdealing with these tumours will be presented separately.

examined to date (Table 1) may be quantitatively removed from the gel surface bythis procedure. The particular sequence of enzyme treatments (i.e. trypsin followedby collagenase) is required for the removal of cells from the gel surface since exposureto collagenase alone for 20 min removes very few surface cells (less than 5 %) and ex-posure to collagenase first and then trypsin removes only as many cells as would beremoved by trypsin alone. Treatment with hyaluronidase and chrondroitinase ABChave not been found to remove cells from the gel surface when used alone or followingtrypsin (data not shown).

Following treatment of the collagen gels with trypsin and collagenase as describedabove, more than 95 % of the gel still remains intact, as estimated by gel thicknessand weight. Cells which have infiltrated into the gel matrix can be recovered in aquantitative manner by completely dissolving the remaining gel with a secondtreatment of collagenase for approximately 5 h. The total cell number is then calcu-

Cell proliferation and migration on collagen 169

100

Ish

10

10

8 _

oa?

2 4 6 8 10 12Days

Fig. 6. The growth and infiltration of BHK cells on 3-dimensional gels of nativecollagen fibres. BHK cells in MEM containing 10% foetal calf serum were plated onthe surface of 3-dimensional collagen gels and the increase in total cell number deter-mined at various times thereafter as described in Materials and methods by dissolutionof the gel with collagenase (Coulter counter method) (#—#) and by direct microscopicexamination (A— A )• The percentage of the total cell number within the 3-dimensionalgel matrix was determined by selective removal of cells from the gel surface (Coultercounter method) (# — 9 ) and by direct microscopic examination (A — A).

lated by adding the number of cells recovered from the gel surface and those withinthe gel matrix, and these data are used to determine the proportion of cells within thegel.

The proportion of cells within the gel matrix can also be determined by directmicroscopic examination. This is simply accomplished by counting the cells on andbelow an area of the gel surface defined by a graticule in the microscope eyepiece.This operation is repeated approximately 20 times at areas of the gel surface selectedat random moving in a straight line across the diameter of the culture dish. Data ob-tained in this manner can be used to calculate the mean + S.D. of the cell number onand below a defined area of the gel surface. In this case, the magnitude of the S.D.is a function of the randomness of cell distribution on the gel surface. Data regardingcell number can be expressed in absolute terms (total cells/dish) by making theappropriate calculations, knowing the magnification of the objective and area of thegraticule field (Materials and methods).

The results obtained by using both of these methods to determine total cell numberand percentage of cells within the gel matrix are shown in Fig. 6. BHK cells wereplated on the surface of 3-dimensional collagen gels and examined at various times

CEL 41

170 S. L. Schor

1 0 r

1

A - T —A-i 1—

20

15 -

10 -S

2 4 6 8 10 12 14 16Days

Fig. 7. The growth and infiltration of human skin fibroblasts on 3-dimensional gelsof native collagen fibres. Human skin fibroblasts (adult, passage 5) in MEM con-taining 15 % foetal calf serum were plated on the surface of 3-dimensional collagengels and the increase in total cell number determined at various times thereafter asdescribed in Materials and methods by dissolution of the gel with collagenase (Coul-ter counter method) ( • — • ) and by direct microscopic examination (A—A). Thepercentage of total cell number within the 3-dimensional gel matrix was determinedby direct microscopic examination (A — A)

during the next 12 days. In spite of the considerable differences in the proceduresused, the results obtained by both methods indicate an exponential rise in total cellnumber to a final density of approximately 1-5 x io6 cells/dish. More germane to thepresent discussion, however, are the data regarding the percentage of cells within thegel matrix. In this case, both methods also give essentially the same results and indi-cate that BHK cells began to move into the gel matrix between days 6 and 8, andthat the percentage of cells within the gel increased in a linear fashion thereafter,resulting in approximately 6% of the total cell number within the gel matrix at day12. Data from a similar experiment with human skin fibroblasts are shown in Fig. 7.Cells were plated on the surface of collagen gels at an initial density of 1-7 x 10* cells/dish. In this case the increase in total cell number was determined by both methods,while the percentage of cells within the gel matrix was determined only by microscopicobservation. Both methods reveal a similar exponential increase in total cell numberuntil a plateau value of approximately 5 x io4 cells/dish was reached after 10 days.Cells began to migrate into the gel 6—10 days after the beginning of the experimentand the percentage of cells within the gel increased in a linear fashion thereafter to avalue of approximately 20% on day 16.

Of the tumour cells examined to date, those of non-epithelial origin may infiltrateinto the gel matrix, while those of epithelial origin appear to be confined to the gel

Cell proliferation and migration on collagen 171

Table 2. The growth and infiltration of Syrian hamster melanoma cells(RPMI-34.60) on ^-dimensional collagen gels

Day

12

Coulter

Total cells( X IO6)

2-564

counter method

% in gel matrix

9022-1

Microscopic

Totalcells ( x io6) %

2-9 ±o-86-i ±1-3

method

, in gel matrix

7-7 ±3-225-4 ±68

Cells in MEM containing 10 % serum were plated on the surface of 3-dimensional collagengels at an initial density of io6 cells/dish and the total cell numbers and percentage of total cellswithin the gel matrix determined 1 and 2 days later as described in Materials and methods bythe ' Coulter counter method' and ' direct microscopic observation'.

surface (Table 1). Quantitative data concerning the infiltration of RPMI-3460Syrian hamster melanoma cells are presented in Table 2. Cells were plated on thesurface of collagen gels at an initial density of ioB cells/dish and the percentage of cellswithin the gel matrix determined 1 and 2 days later by both methods. These cellsshow a greater degree of infiltration than either the BHK cells or human skin fibro-blasts, with approximately 20% of the melanoma cells within the gel matrix after 2days of growth.

DISCUSSION

The main purpose of this communication is to present quantitative data regardingcell proliferation and migration on collagen substrata in vitro. In spite of the fact thatcells have been cultured on collagen substrata for many years (Ehrmann & Gey,1956), this type of quantitative information has not previously been reported.

Before discussing the results, it is important to emphasize that the nature of thecollagen substratum is particularly important and must be given careful consideration.Various types of collagen have been identified in vivo and characterized in terms ofdifferences in biochemical parameters and tissue distribution (Grant & Jackson,1976; Miller, 1977). These different types of collagen can be used to form substrataconsisting of supramolecular complexes of collagen (Ehrmann & Gey, 1965; Klebe,1.975; Rubin et al. 1978; Elsdale & Bard, 1972; Schor & Court, 1979) the detailedstructure of which may vary as a function of pH, ionic strength, etc. Chick corneaepithelial cells have been reported to respond in an identical manner to differenttypes of collagen substrata in terms of increased synthesis of collagen and glycosamino-glycans (Meier & Day 1974), while the differentiation and adhesive characteristicsof other cells display a pronounced specificity for both the type of collagen (Murrayet al. 1979) and its molecular organization within the substratum (Schor & Court,1979; Grinnell & Minter 1978). In the work reported here, type 1 collagen extractedfrom the rat tail tendons was used to make collagen films and 3-dimensional gels ofnative collagen fibres; the molecular organization of collagen within these substrata

172 S. L. Schor

has been discussed previously (Schor & Court, 1979), and subsequently confirmedby observation with the electron microscope (Schor & Allen, in preparation).

Type 1 collagen is widely distributed in the organism, being present as interstitialfibres in dermis and other mesenchymal tissues. The behaviour (proliferation andmigration) of human skin fibroblasts on these collagen substrata is therefore of par-ticular interest, since these cells are embedded in an extracellular matrix containingtype 1 collagen fibres in vivo. Epithelial cells on the other hand are often in contactwith a basement membrane containing type IV collagen, but the behaviour of thesecells on the type 1 collagen substrata used here is still of interest since (a) type 1 colla-gen substrata have been shown to influence the expression of a differentiated pheno-type in a number of epithelial cells in vitro, including liver parenchymal cells (Michalo-poulos & Pitot, 1975), cornea epithelial cells (Meier & Hay, 1975) and mammaryepithelial cells (Emermann & Pitelka, 1973), and (b) epithelial cells may proliferatein or migrate through extracellular matrices containing type 1 collagen fibres duringvarious pathological conditions, such as wound healing and tumour cell invasion(Winter, 1972; Fidler, Gersten & Hart, 1978). Experiments similar to those reportedhere are currently in progress using substrata prepared from other types of collagen.

Quantitative data have been presented concerning the proliferation of HeLa cells,BHK cells and human skin fibroblasts on collagen films, on the surface of collagengels and within the 3-dimensional gel matrix. Cells were grown in medium containingconcentrations of foetal calf serum required for optimal growth on plastic tissue culturedishes. HeLa cells were found to proliferate at approximately the same rate on allsubstrata, while human skin fibroblasts cultured within the 3-dimensional gel matrixshowed a significant lag period before growth commenced. The reasons for theslower growth of fibroblasts within the gel matrix are not known, but may result fromseveral factors, including the availability of nutrients and the fact that cells within thegel are embedded in an homogeneous 3-dimensional environment, while cells on thesurface of the gels (or collagen films and plastic dishes) are growing on a 2-dimensionalsurface, with the resultant changes in cellular physiology that this may induce. Theresults of further experiments dealing with these questions will be reported separately.At this point it is worthwhile to note that under normal conditions in vivo, skinfibroblasts are slowly growing cells embedded in a 3-dimensional collagen matrLx inthe presence of interstitial fluid (not serum).

Data have also been presented concerning the migration of cells on collagen sub-strata. The migration of cells on the surface of collagen films and gels was assessed bythe distance between daughter cells in individual colonies after several days of growth.These data indicate that HeLa cells migrate considerably more on the surface ofcollagen substrata than on plastic tissue culture dishes. A similar approach has beenused by Ali & Hynes (1978) in an investigation of the effects of fibronectin on cellmigration. These authors report that cell-surface-derived fibronectin stimulates themigratory behaviour of fibroblasts on plastic tissue culture dishes. In view of the factthat fibronectin binds specifically to collagen (Yamada & Olden, 1978), it is possiblethat the differences in the migratory behaviour of HeLa cells on the different substratareported here (in medium containing the same amount of fibronectin) may be due to

Cell proliferation and migration on collagen 173

the preferential binding of fibronectin to collagen and the subsequent migration of thecells on the fibronectin-collagen complex.

The collagen gels can also be used to assess the ability of cells to migrate (infiltrate)into the 3-dimensional gel matrix. Tickle, Crawley & Goodman (1978a, b) examinedthe migratory behaviour of a number of different cell types implanted into thedeveloping chick wing bud and report that mesenchymal cells (e.g. BHK, PyBHK)and various tumour cells (e.g. neuroblastoma) migrated freely into the surroundinghost mesenchymal tissue, whereas both normal and tumour cells of epithelial origindid not. The data presented by these authors are in agreement with the ability of thesecells to infiltrate into the 3-dimensional collagen gel in vitro reported here (Table 1).

It is apparent that the ability of cells to migrate on the gel surface and infiltrate intothe 3-dimensional gel matrix are distinct attributes of cell behaviour, since HeLacells do not move into the gel matrix (Table 1), in spite of the fact that these cellsdemonstrate increased migratory activity on the surface of the gels compared toplastic dishes (Fig. 4) and can proliferate rapidly within the gel matrix (Fig. 1). Therelationship between cell migration and proliferation will be described in greaterdetail elsewhere (Schor, in preparation).

Other experimental systems have been developed to obtain quantitative informationconcerning cell migration (Burk, 1973; Harrington & Stastny, 1973), but thesegenerally employ artificial substrata (such as plastic tissue culture dishes) on whichcell migratory behaviour may be quite different from that on the types of substratanormally encountered in vivo. The gels of native collagen fibres described hereshould provide a more suitable substratum for the study of cell migratory behaviour,a conclusion supported by the similarities in cell behaviour on these gels in vitroand that observed both in the collagen matrix of the developing chick cornea in vivo(Bard & Hay, 1975) and on isolated basement membranes in vitro (Overton, 1977).The migration of cells on artificial substrata in vitro is known to be influenced by anumber of factors, including proteases (Ossowski, Quigley & Reich, 1975; Varani,Orr & Ward, 1979), fibronectin (AH & Hynes, 1978) and serum factors (Lipton,Klinger, Paul & Holley, 1971; Burk, 1973). Cells which infiltrate into the collagen gelmatrix in vitro (e.g. human skin fibroblasts and RPMI-3460 melanoma cells) have beenmaintained in culture for periods of up to one month without visible changes in gelvolume or stability, thus making it unlikely that the movement of cells into the gel isaccompanied by a significant amount of fibre lysis due to the production of collagenase.Further data regarding the possibility of more limited collagenolysis (using 3H-labelled collagen), as well as the roles played by fibronectin, serum factors and thecytoskeletal system in cell infiltration, will be presented separately (Schor, in pre-paration).

The work described here is part of a long-term study of tumour cell invasion andmetastasis. Clearly these 2 processes are complex biological phenomena, showingconsiderable variation between different types of tumours and involving a multipleof tumour-host interactions (Fidler et al. 1978). One particular aspect of these tumour-host interactions which should be considered is the ability of the tumour cells tomigrate or infiltrate through the collagen fibres of the extracellular matrix (Strauli &

174 S. L. Schor

Weiss, 1977). Of the tumour cells examined to date, those of epithelial origin do notinfiltrate into the 3-dimensional collagen matrix in vitro, while those of non-epithelialorigin may do so. The differences in the infiltrative behaviour of these tumour cellson the collagen gels in vitro may reflect their different modes of invasive growth invivo (Willis, 1973) and is the subject of current investigation.

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{Received 16 July 1979)