Collagenase

PRELIMINARY

NOTES

Collagenase Its effectiveness as a dispersing agent for embryonic chick thyroid and heart S. R. H I L F E R 1 and J. M. BROWN, 2 IDepartment of Biology, Temple University, Philadelphia, Pa 19122, and ~Medical Service Corps, Department of Physiology, Dental School, University of Maryland, Baltimore, Md 21201, USA

Summary The effectiveness of tetraphenylboron, pronase and collagenase was tested on embryonic chick thyroid and heart. The relative dispersing efficiency was judged by number of cells released and their viability in monolayer culture. Tetraphenylboron was not effective because it killed the cells. Pronase and collagenase both released large numbers of cells but collagenase treatment resulted in greater survival. Tests of proteolytic activity of collagenase as a means of explaining the results revealed that all commercial preparations contain some activity under the conditions used for dissociation.

Collagenase has become one of the most commonly used dissociating agents in the preparation of cell suspensions for in vitro cultivation [1, 11] because it seems to cause less cellular damage than other enzymes or chelating agents. However, the basis for this gentler effect is not known. These experiments were performed in order to compare treatment with collagenase, an enzyme with a highly specific substrate: pronase, an enzyme with broad substrate specificity: and tetraphenylboron (TPB), a chelating agent preferential for potassium, with respect to both efficiency of cell dispersal and cell viability. Thyroid and heart ventricle were chosen as the cell source for comparison with work previously done in our laboratory [4] using trypsin and ethylenediaminetetraacetate (EDTA). Thyroids and heart ventricles of Rhode Island Red embryos were removed to and Exptl Cell Res 65

cleaned in Tyrode saline. Samples of approximately equal size consisted of 40 thyroids or 4 ventricles of 8-day embryos, or 10 thyroids or 3 ventricles from 14- and 16-day embryos. The intact thyroid or cubed ventricular tissue was first rinsed in potassium-free Tyrode for 20 min in a Dubnoff shaker at 37~ at a speed of 40 strokes/min. After transfer to dissociating medium, the tissue was incubated with shaking for an additional 30 rain. Either collagenase (0.25%), pronase (0.25%), or TPB (10 -a M for 8-day or 10-2 for 14- and 16-day) dissolved in potassiumfree Tyrode was used to effect dissociation. The samples were next spun at 3 200 rpm in a clinical centrifuge, resuspended in fresh potassium-free Tyrode's, and mechanically separated by vigorous pipetting with a Pasteur pipette. After the debris had settled, the suspended cells were transferred to a fresh centrifuge tube and the volume adjusted to 10ml. Duplicate cell counts were made with a haemocytometer. Clumps were recorded as such but not analysed for cell number. Viability was tested by plating cells in Hanks F12 supplemented with H or HI embryo extract and 5% fetal calf serum [2, 10], in Falcon culture flasks. Medium was changed on alternate days and the cultures were examined for attachment, morphology, and general growth characteristics. Proteolytic activities of collagenase (CalBiochem, Sigma, Nutritional Biochemical), collagenase fractions A and B (Worthington), and pronase (CalBiochem) were compared by the casein-Folin method as used by the Kaken Chemical Co. for the standardization of pronase, except that all reactions were

Collagenase dissociation 247 Table 1. Efficiency of dissociation of thyroid and heart by tetraphenylboron, pronase, and

collagenase

Embryonic age (days)

TPB

Pronase

Collagenase

No. cells released ( • 104) a

No. cells released ( x 104)

No. cells released ( • 104)

Viability b

Viability

Viability

8 Thyroid Heart

5.16 32.00

0 (6)

15.90 63.40

• (8)

10.33 53.40

+ + (8)

14 Thyroid Heart

16.22 34.80

0 (8)

45.50 153.00

+_ (8)

66.25 131.40

+ + (6)

16 Thyroid Heart

41.50 125.00

0 (6)

85.80 206.00

_+ (8)

87.55 237.00

+ + (8)

a See text for conditions of dissociation. Number in parentheses indicates number of samples of each cell type tested. 0, no attachment; ___, some attachment and little growth; +, attachment and growth; + +, attachment and prolific growth. performed at 37~ instead of 40~ and potassium-free Tyrode's was used in place of their buffer. Comparison of the number of cells released by the three agents (table 1) reveals the relative inefficiency of TPB as a dispersing agent. The yield was consistently only onethird to one-half that of the two enzymes. On the other hand, there was little difference between pronase and coUagenase with respect to the number of cells released. The same pattern of response was obtained for both heart and thyroid. The age of the organ had a significant effect upon the effectiveness of any one of the agents, although it was more pronounced with thyroid than with heart. The difference seems to lie in a disproportionate number of cells released from the 8-day heart and may reflect the difference in maturity of the two organs at that embryonic age. The studies on viability (table 1) were made in a nutrient medium selected to give maximal chance of survival. Therefore, the lack of maintenance in any culture can be taken as a sign of cellular damage. Cells which were released in TPB, regardless of age, showed little growth in culture, but the

few cells which did attach in the culture flasks appeared completely normal under phase optics. The suspensions before plating contained many objects which appeared to be either isolated nuclei or nuclei with only a thin covering of cytoplasm. Tetraphenylboron-treated cells became excessively sticky, a tendency which was not reduced by resuspension in fresh medium or even additional medium changes. Those cells which did not attach in the culture flasks had dense nuclei and many surface blebs. When these "floaters" were serially passed through several changes of fresh or conditioned medium they still did not attach and grow. Tissues dissociated in pronase yielded large numbers of single cells, but when placed in culture, many of the cells did not attach and, as a result, the survival in culture was generally poor. The thyroid cells formed broad, flattened sheets. There was evidence of piling up of cells and also granular inclusions in many of the cells. The heart ventricle cells showed similar growth characteristics, but were spindled-shaped. Serially passed "floaters" of pronase-treated cells attached and grew, whereas in TPB they did not. Collagenase-treated cells consistently grew Exptl Cell Res 65

248

S. R. Hiller & J. M. Brown

1.400

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~

et

9/

o0oI/";/

o

I

I

I

I

|

0,05

0.10

0.15

O.ZO

0,25

4,'I/4

Fig. 1. Abscissa: enzyme concentration ordinate: optical density.

,I,

2.5

(mg/ml);

Comparison of proteolytic acivity of collagenase preparations. Intensity developed with 1% casein was red at 660 M, l , pronase, Calbiochem; A, collagenase, Sigma; e , collagenase, Nutritional; 9 collagenase, Worthington, CLSPA & CLSPB.

well in culture regardless of the cell source or the age of the donor. They attached rapidly and were spreading within 30 min of the time of inoculation. The thyroid cells, while exhibiting slightly different morphological characteristics from the pronase-separated cells, were also epithelial in appearance. Heart cells separated with collagenase exhibited no noticeable morphological differences from those separated with pronase. Since the removal of a surface protein may be one method by which dissociation is accomplished, the three agents were tested for proteolytic activity by measuring their effect on casein. The same conditions were used under which the cell dissociations were performed so that a measure of activity under these conditions could be obtained. These results are summarized in fig. 1. Tetraphenylboron, a chelating compound, exhibited no proteolytic activity in concentration of 10-2M or 10-3M. Collagenase, howExptl Cell Res 65

ever, did demonstrate proteolytic activity as did pronase. Pronase is a proteolytic enzyme with a broad substrate specificity and thus could be expected to show a high activity. Pure collagenase shows no activity on casein [6, 7] and thus the collagenase used in this study must have contained an additional component or components which exhibited proteolytic activity. Since the proteolytic activity of all enzyme preparations was so high at 2.5 mg/ml (0.25 %), concentration curves were plotted (fig. 1). Sigma collagenase had 41% as much proteolytic activity as pronase, and Nutritional Biochem. collagenase 30% as much, at 0.02 mg/ml. The purified Worthington samples did not contain detectable proteolytic activity even at 0.25 mg/ml, but at concentrations used for dissociation their activity was appreciable. It should be noted that both purified and crude collagenases were effective for cell dissociation. When solutions of collagenase and pronase were compared by acrylamide gel electrophoresis according to the method of Fleischmajer & Krol [3], it was clear that there were many components in both purified and crude samples. Furthermore, a comparision of enzyme preparations from various commercial sources indicated that all contain more than one component. It is not known which of these components contain the proteolytic activity. The results suggest that the effectiveness of collagenase for cell dispersal is not necessarily related only to the degradation of collagen. This enzyme preparation is effective in separating epithelial cells, which have no collagen fibrils along their lateral borders, as well as the heart cells, which are surrounded by fibers. The demonstrable proteolytic activity of all commercial collagenase preparations which have been tested also supports this conclusion. In fact, it has been known for some time that it is extremely difficult to

Filaments in the cleavage furrow rid collagenase of all proteolytic activity. At least one contaminant, clostridiopeptidase B, has been shown to have properties similar to trypsin [8]. It has also been shown recently that collagenase preparations contain variable amounts of glycopeptidase activity [12]. Perhaps the effectiveness of collagenase as well as the superior properties of the cruder trypsin preparations, then, lies in the presence of their contaminants. The three agents show significant differences in the ability to dissociate the two tissues that were tested. Since the purpose of dispersing techniques is to provide large numbers of viable cells, both the number of cells released and the extent of damage are important factors in judging the efficiency of any method. Although there was some difference in the performance of the two enzymes, they both proved to be far superior to the chelating agent. The original work with TPB suggested excellent efficiency when used for the isolation of cells from adult mouse liver [9]. More recently, this agent has been reported to cause extensive damage to liver ultrastructure [5]. The limited number of cells released from embryonic chick thyroid and heart and their total inviability is also a reflection of the extensive damage to cell structure which is caused by this agent. The effects upon cytoplasmic and nuclear fine structure will be described in a separate publication. The difference in effectiveness of the two enzymes, on the other hand, is related to cell survival rather than ability to disperse, since both provided almost equivalent numbers of cells. Previously, the method of choice for thyroid dissociation has been a combination of trypsin and EDTA. However, trypsin by itself does not separate follicle cells of the older gland but subsequent exposure to EDTA, although it does release the epithelial cells, causes cytoplasmic damage [4]. Therefore, collagenase is the most 17 - 711804

2 49

effective agent for dissociation that has been tried so far. This work was supported in part by grant GB3925 from the National Science Foundation. Submitted in partial fulfillment of the requirements for the M. A. Degree at Temple University. The views expressed herein are those of the authors and do not necessarily reflect the views of the United States Army or the Department of Defense.

REFERENCES 1. Cahn, R D, Coon, H G & Cahn, M B, Methods in developmental biology (ed F H Wilt & N K Wessells) p. 493. Crowell, New York (1967). 2. Coon, H G & Cahn, R D, Science 153 (1966) 116. 3. Fleischmajer, R & Krol, S, Proc soc exptl biol med 126 (1967) 252. 4. Hilfer, S R & Hilfer, E K, J morphol 119 (1966) 217. 5. Kerkof, P R, Smith, S, Gagn6, H T, Pitelka, D R & Abraham, S, Exptl cell res 58 (1969) 445. 6. Mandl, I, Advances in enzymology(ed F F Nord) p. 163. Interscience, New York (1961). 7. Mandl, I, MacLennan, J D & Howes, E L, J clin invest 32 (1953) 1323. 8. Mitchell, W M & Harrington, W R, J biol chem 243 (1968) 4683. 9. Rappaport, C & Howze, G B, Proc soc exptl biol med 121 (1966) 1010. 10. Spooner, B S, J cell physiol 75 (1970) 33. 11. Steinberg, M S, Methods in developmental biology (ed F H Wilt & N K Wessells) p. 565. Crowell, New York (1967). 12. Wessells, N K & Bernfield, M R, Symp soc developmental biol (1970). In press. Received February 9, 1970 Revised version received December 8, 1970

Actin-like filaments in the cleavage furrow of newt egg M. M. PERRY, H. A. JOHN and N. S. T. THOMAS, Institute of Animal Genetics, University of Edinburgh, Scotland There is a growing body of evidence that cytoplasmic filaments are implicated in cell cleavage. In the fertilized newt egg, a band of parallel filaments lying beneath the plasma membrane and oriented in the plane of the first cleavage furrow has been reported by Selman & Perry [17]. Similarly situated filaments have been described in the dividing Exptl Cell Res 65