Tissue Culture: Methods and Applications

Tissue Culture: Methods and Applications presents an overview of the procedures for working with cells in culture and for using them in a wide variety of scientific disciplines. The book discusses primary tissue dissociation; the preparation of primary cultures; cell harvesting; and replicate culture methods. The text also describes protocols on single cell isolations and cloning; perfusion and mass culture techniques; cell propagation on miscellaneous culture supports; and the evaluation of culture dynamics. The recent techniques facilitating microscopic observation of cells; cell hybridization; and virus propagation and assay are also encompassed. The book further tackles the production of hormones and intercellular substances; the diagnosis and understanding of disease; as well as quality control measures. Scientists and professionals interested in methodology per se will find the book invaluable.

• Language: English
• Publisher: Academic Press
• Release date: Dec 2, 2012
• ISBN: 9780323142076

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JR.

SECTION I

PRIMARY TISSUE DISSOCIATION

Introduction to Primary Tissue Dissociation

Editors’ Comments

The dissociation of organized tissues into single cell suspensions requires a dissolution or weakening of the extracellular matrix (stroma). The choice of method thus depends on the composition of the stromal material. Other parameters, however, must be considered, namely, the quality and quantity of cells desired. There is little doubt that the use of hydrolytic enzymes to effect dispersion of cells alters the cellular exterior. Such enzymes are used in most cases not only because there appears to be no substitute for them, but also because with their use the cells appear to repair minor membrane damage. To obtain quality cells, it is apparent that a minimum of mechanical trauma and exposure time to the hydrolytic enzymes must be achieved. Larger quantities of cells with a lower viable population can be obtained, of course, by longer exposure times.

The methods described in this section refer to specific tissues, and the investigator should not infer that they can successfully be used with every tissue. For example, it is illustrated that the saline vehicle which is satisfactory for preparations of cells from marine fish tissues is different in composition from that used with tissues of other animals, including freshwater fish.

This section also includes two methods for nonenzymatic dissociation or, perhaps more appropriate, fractionation of cell types. Both make use of the fact that certain cell types differ in the degree to which they attach to various surfaces; these novel techniques might serve as the basis for developing cell separation procedures for other tissues composed of heterogeneous cell populations.

The reader is referred also to other descriptions of cell preparations elsewhere in the book, particularly Section II in which dissociation of specific tissues in preparing primary cultures is discussed, e.g., for separating cardiac myocardial and endothelial cells.

CHAPTER 1

Pronase

Ralph B.L. Gwatkin

Publisher Summary

This chapter describes the use of pronase in tissue culture. Pronase, an enzyme preparation from Streptomyces griseus, consists of a mixture of neutral and alkaline proteases, aminopeptidases, and carboxypeptidases. It has been used to remove the zona pellucida of mammalian eggs, to liberate chondrocytes from cartilage prior to freezing, and in tissue culture. Applications of Pronase in tissue culture include the dissociation of chicken and mouse embryo cells prior to cultivation and subcultivation. Pronase acts much more rapidly than trypsin, and unlike trypsin, it yields cell suspensions without large clumps of cells. It is also superior to trypsin in digesting dead cells and cellular debris. When Pronase is used to subculture cell monolayers obtained from mouse embryo cells, primary cultures of human diploid fibroblasts, and epithelial cells derived from monkey kidney tissue the cells are brought rapidly into suspension. However, certain continuous cell lines, for example, HeLa and KB are removed in flakes, indicating incomplete dispersion. The chapter describes the procedure for making primary cell cultures from 14-day mouse.

Pronase, an enzyme preparation from Streptomyces griseus, consists of a mixture of neutral and alkaline proteases, aminopeptidases, and carboxypeptidases.¹ It has been used to remove the zona pellucida of mammalian eggs,²–⁴ to liberate chondrocytes from cartilage prior to freezing,⁵ and in tissue culture.

Applications of Pronase in tissue culture include the dissociation of chicken and mouse embryo cells prior to cultivation and subcultivation.⁶,⁷ Pronase acts much more rapidly than trypsin and, unlike trypsin, yields cell suspensions without large clumps of cells⁷ (see Fig. 1). It is also superior to trypsin in digesting dead cells and cellular debris.⁸ When Pronase is used to subculture cell monolayers obtained from mouse embryo cells,⁷ primary cultures of human diploid fibroblasts,⁹–¹¹ and epithelial cells derived from monkey kidney tissue¹¹ the cells are brought rapidly into suspension. However, certain continuous cell lines, e.g., HeLa and KB are removed in flakes, indicating incomplete dispersion.¹¹,¹² The reason for the relative ineffectiveness of Pronase with these cell lines is not known. Prolonged subcultivation of diploid human fibroblasts with Pronase was found to have no detectable effect on karyotype or on their growth potential.⁹,¹⁰ However, since potent inhibitors of Pronase comparable to antitrypsins are not present in serum, the cells should be washed free of residual enzyme before initiating new cultures.¹¹

Fig. 1 Cells released after digestion of 13-day mouse embryo tissue with 0.25% Pronase (left) and 0.25% trypsin (right). Note that Pronase yields a monodisperse cell suspension, while trypsin leaves large clumps of cells intact.

PRIMARY CELL CULTURES FROM 14-DAY MOUSE EMBRYOS

Pronase Solution (0.25%).

Add 1.25 g standard Pronase (Cat. No. 53702, Calbiochem Corp., 10933 N. Torrey Pines Road, La Jolla, California 92037) to 500 ml phosphate-buffered saline (PBS).¹³ Mix and allow to stand for 30 minutes at room temperature. Centrifuge (100 g for 10 minutes) to remove undissolved material,¹⁴ then sterilize by filtration (Millipore GS, 0.22 pore size). Store 50-ml aliquots frozen.

Dissociation of the Tissue.

Kill mice on thirteenth to fourteenth day of pregnancy (12–13 days after a copulation plug is found in the vagina) by quickly stretching their necks. Swab abdomens with 70% ethanol, open abdomen, and remove uterine horns to a Petri dish containing PBS. Slit open uterine horns to obtain embryos. Wash them thoroughly in PBS and place 10 embryos in a 10-ml hypodermic syringe (without a needle). Force embryos through the syringe into a 125-ml Erlenmeyer flask containing 50 ml 0.25% Pronase in PBS (see above). This single passage through the orifice of the syringe reduces the embryos to a pulp with minimal damage to their constituent cells. Drop a sterile magnetic bar into the flask, place the flask on a magnetic stirrer, and stir slowly at room temperature for 2 hours. Add 5 ml sterile calf serum; stir again for 5 minutes and pipette up and down several times. Cells now appear as seen on left of Fig. 1. Decant into a centrifuge tube (50 ml) and centrifuge cells at 100 g for 12 minutes. Pour off the supernatant and resuspend the cells in 20 ml growth medium (Eagle’s Minimal Essential Medium¹⁵ supplemented with 10% calf serum). Centrifuge and resuspend the cells twice to remove Pronase.

Primary Cultures.

Add 5-ml aliquots of the cell suspension (approximately 2 × 10⁸ cells) to each of four plastic culture flasks (75 cm²). These are prepared beforehand by adding 10 ml growth medium to each and gassing them with 5% CO2 in air. Incubate the cultures at 37°C. After 24 hours change the medium to remove cellular debris. Monolayers of cells are usually complete and ready for subcultivation after 5 days.

Subcultivation.

Pour off medium and wash monolayers once with PBS. Add 10 ml Pronase solution (diluted 1:5 with PBS to give a final Pronase concentration of 0.05%). Cells become detached as single cells in 3–5 minutes. Add 1 ml calf serum, pipette up and down several times, and transfer to a 20-ml centrifuge tube. Centrifuge and resuspend in growth medium twice to remove Pronase as already described. Finally, resuspend in growth medium and use this suspension to prepare new cultures, e.g., monolayers for virus titrations.¹⁶

References

1. Narahashi, Y., Shibuya, K., Yanagita, M. J. Biochem. (Tokyo). 1968; 64:427.

2. Mintz, B. Science. 1962; 138:594.

3. Gwatkin, R.B.L. J. Reprod. Fert. 1963; 6:325.

4. Gwatkin, R.B.L. J. Reprod. Fert. 1964; 7:99.

5. Smith, A.U. Nature (London). 1965; 205:782.

6. Wilson, B.W., Lau, T.L. Proc. Soc. Exp. Biol. Med. 1963; 114:649.

7. Gwatkin, R.B.L., Thomson, J.L. Nature (London). 1964; 201:1242.

8. Stewart, C.C., Ingram, M. Blood. 1967; 29:628.

9. Sullivan, J.C., Schafer, I.A. Exp. Cell Res. 1966; 43:676.

10. Weinstein, D. Exp. Cell Res. 1966; 43:234.

11. Foley, J.F., Aftonomos, B. J. Cell. Physiol. 1970; 75:159.

12. Kahn, J., Ashwood-Smith, M.J., Robinson, D.M. Exp. Cell Res. 1965; 40:445.

13. Dulbecco, R., Vogt, M. J. Exp. Med. 1954; 99:167.

14. A completely soluble Pronase (Cat. No. 537011) and a nuclease-free Pronase (Cat. No. 537088) are now available from Calbiochem Corp. Further study is needed to determine whether these preparations have any advantages over standard Pronase.

15. Eagle, H. Science. 1959; 130:432.

16. Gwatkin, R.B.L. Fert. Steril. 1966; 17:411.

CHAPTER 2

Trypsin

A. Mammalian Tissues

Charles Shipman, Jr.

Publisher Summary

This chapter discusses the use of trypsin in cultures of mammalian tissues. Trypsin is a pancreatic proteolytic enzyme that preferentially catalyzes the hydrolysis of peptide bonds between the carboxy group of arginine or lysine and the amino group of another amino acid. Because of the inherent problems in batch methods of trypsinization, continuous operating systems of varying degrees of sophistication have been developed. The continuous methods of trypsinization obviate to some degree over-digestion because of the prolonged mean residence time (MRT) of monodisperse cells in the enzyme solution. Those devices that rely upon a sintered-glass filter for cell sizing are easily clogged, whereas other devices do not minimize MRT or are so mechanically complex that their construction is not feasible in most laboratories. The chapter also describes the operations of a simple glass device wherein enzymatically released monodisperse cells can be separated and isolated from tissue fragments by the means of a discontinuous fluid velocity gradient. It describes the method for the preparation of 0.25% trypsin in HEPES-buffered saline (HBS). The chapter also discusses the use of the continuous flow velocity gradient trypsinizing cylinder.

Trypsin is a pancreatic proteolytic enzyme which preferentially catalyzes the hydrolysis of peptide bonds between the carboxy group of arginine or lysine and the amino group of another amino acid. For an extensive review of this enzyme see Desnuelle.¹

The enzymatic release of monodisperse cells from tissue fragments historically has its origin with the early work of Rous and Jones.² These monodisperse living cells can be obtained from animal tissues by trypsinization because the enzyme differentially degrades the protein matrix which binds the cells in the tissue and releases these cells in suspension before they are seriously damaged in the process.

Discontinuous batch methods were developed later³–⁶ and are still in widespread use. Because of the inherent problems in batch methods of trypsinization, continuous operating systems of varying degrees of sophistication have been developed.⁷–⁹ The continuous methods of trypsinization obviate to some degree overdigestion due to prolonged mean residence time (MRT) of monodisperse cells in the enzyme solution. Those devices which rely upon a sintered-glass filter for cell sizing are easily clogged⁷,⁹,¹⁰ whereas other devices do not minimize MRT⁸ or are so mechanically complex that their construction is not feasible in most laboratories.¹¹

Recently a simple glass device has been developed wherein enzymatically released monodisperse cells can be separated and isolated from tissue fragments by means of a discontinuous fluid velocity gradient.¹² The operation of this unit will be described in this subsection. Discontinuous batch methods of trypsinization are described in other sections.

PREPARATION OF 0.25% TRYPSIN IN HEPES-BUFFERED SALINE (HBS)

Since the pH of a balanced salt solution buffered by HEPES (see Section XIV) is unaffected by the composition of the overlaying atmosphere there seems little reason to use a NaHCO3-buffered salt solution to dissolve trypsin.

The formula of a HEPES-buffered salt solution¹³ suitable for the compounding of a trypsin solution is given in Table I.

TABLE I

Formulation of 0.01 M HEPES-Buffered Saline (HBS)a

aThe tonicity of this balanced salt solution is approximately 290 mOsm/kg.

To prepare 1 liter of 0.25% trypsin, 2.5 g of commercially available tiypsin (1:250)¹⁴ are added to 1 liter of HBS and stirred overnight at 4°C. The following day the solution is warmed to room temperature, the pH readjusted to 7.4 (corresponding to approximately 7.2 at 37°C), and sterilized by means of membrane filtration. If larger quantities are prepared it is often desirable to prefilter the solution through a 0.8 µm (mean pore diameter) filter so as not to clog the 0.2 μm sterilizing filter.

USE OF THE CONTINUOUS FLOW VELOCITY GRADIENT TRYPSINIZING CYLINDER¹²,¹⁵

The device consists of a single Pyrex reactor which can be constructed with or without a water jacket (Fig. 1). The inner cylinder is 38 mm (OD), whereas the water jacket is 160 mm (OD). Reactor volume is approximately 75 ml.

Fig. 1 Drawing of the automatic trypsinizing device. A, Water jacket; B, inlet, water jacket; C, outlet, water jacket; D, trypsinizing chamber with four flutes; E, inlet, trypsinizing chamber (connected via regulating valve to trypsin reservoir); F, spillover, trypsinizing chamber. From Shipman and Smith.¹²

Operationally, three velocity patterns of fluid movement are involved. In the region below the flutes, the minced tissue fragments are suspended in a vortex by the action of the magnetic stir bar. In the fluted region, aggregates of cells are released by the combined action of the turbulence and the enzymatic digestion. Above the flutes, there is neither a vortex nor turbulence but only an upward component of velocity. Monodisperse cells occupy this upper region of the vessel prior to their egress from the chamber. It is the small volume (approximately 10 ml) and the upward velocity component which allows one to achieve a minimum MRT.

The unit is normally sterilized with inlet and outlet hoses attached and a magnetic stir bar in the inner cylinder. After sterilization the chamber is aseptically connected to a reservoir of trypsin-HBS and to a delivery vessel for the collection of monodisperse cells. To stop the action of the trypsin, the receiving vessel contains a small amount of serum and is surrounded by crushed ice.

Tissue fragments prepared using a mincing tube and stainless steel barber’s shears are transferred in HBS to the trypsinizing chamber by means of a large orifice volumetric pipette.¹⁵ The magnetic stirring device is turned on and water at 37°C is circulated in the jacket. The flow of trypsin is now started from the reservoir. A Teflon stopcock at the reservoir is used to control flow rate. Alternatively, flow rate can be controlled by using a peristaltic pump. The speed of the stir bar and the flow rate of the trypsin must be empirically determined and are to a large extent dependent upon the type of tissue being trypsinized.

The automatic device has been used with monkey kidney, rabbit kidney, puppy salivary gland, and chick embryo tissues. It has also been utilized to free secretory cells from human thyroid tissue. Cell viability, as measured by trypan blue dye exclusion, and total yield of cells were always as high and often higher than using discontinuous batch methods of trypsinization.

References

1. Desnuelle, P.Boyer, P.D., Lardy, H., Myrback, K., eds. The Enzymes; 4. Academic Press, New York, 1960:119.

2. Rous, P., Jones, F.S. J. Exp. Med. 1916; 23:549.

3. Bodian, D. Virology. 1956; 2:575.

4. Dulbecco, R., Vogt, M. J. Exp. Med. 1954; 99:167.

5. Melnick, J.L., Rappaport, C., Banker, D.D., Bhatt, P.N. Proc. Soc. Exp. Biol. Med. 1955; 88:676.

6. Youngner, J.S. Proc. Soc. Exp. Biol. Med. 1954; 85:202.

7. Barski, G. Ann. Inst. Pasteur (Paris). 1956; 91:103.

8. Bishop, L.W.J., Smith, M.K., Beale, A.J. Virology. 1960; 10:280.

9. Rappaport, C. Bull. WHO. 1956; 14:147.

10. Nicol, L., Girard, O., Corvazier, R., Cheyroux, M., Reculard, P., Sizaret, P. Ann. Inst. Pasteur (Paris). 1960; 98:149.

11. Gori, G.B. Appl. Microbiol. 1964; 12:115.

12. Shipman, C., Jr., Smith, D.F. Appl. Microbiol. 1972; 23:188.

13. Shipman, C., Jr. Proc. Soc. Exp. Biol. Med. 1969; 130:305.

14. 1:250 = One part of trypsin will convert 250 parts of casein to protease, peptones, and amino acids under the conditions of the N.F. assay for pancreatin.

15. Available from Bellco Glass Inc., 340 Edrudo Road, Vineland, New Jersey 08360.

CHAPTER 2

Trypsin

B. High Yield Method for Kidney Tissue

Héctor Montes de Oca

Publisher Summary

This chapter presents a high yield method for kidney tissue, employing trypsin and disodium ethylenediaminetetraacetic acid (disodium EDTA) as the dispersing agents. The advantages of this method, as compared with other standard dispersion methods, are marked as increase in the cell yield, reduction in the time required to accomplish complete dissociation of the tissue, and production of a culture with a uniform and clean monolayer that facilitates detection of viral cytopathogenic effect. Description of the present technique is based on the work with simian, rabbit, and human kidneys. In the method described in the chapter, the animal kidneys are obtained from animals anesthetized with Pentothal sodium and killed by exsanguination. Human kidneys are obtained from infant cadavers under sterile conditions. The results obtained from the method, using different dispersing agents, are tabulated in the chapter. The higher percentage of dead cells in the Youngner’s method is because of the centrifugation performed in a conical tube, with all of the dispersed cells, to determine their total volume. When this step is omitted and instead, the cell counts are performed, the number of dead cells is reduced by approximately one-half.

The method to be described here was reported in part elsewhere¹,² and is a combined method employing trypsin and disodium ethylenediaminetetraacetic acid (disodium EDTA) as the dispersing agents. The advantages of this method, as compared with other standard dispersion methods, are marked increase in the cell yield, reduction in the time required to accomplish complete dissociation of the tissue, and production of a culture with a uniform and clean monolayer that facilitates detection of viral cytopathogenic effect.

SOURCE OF TISSUE

Description of the present technique is based on the work with simian, rabbit, and human kidneys. The animal kidneys are obtained from animals anesthetized with Pentothal sodium and killed by exsanguination. Human kidneys are obtained from infant cadavers under sterile conditions. The kidneys are aseptically removed and placed in Hanks’ Balanced Salt Solution with 0.5% (w/v) lactalbumin hydrolysate, 2% (v/v) calf serum, 200 units of penicillin, and 200 μg of streptomycin per milliliter or, preferably, in CMRL 1415 ATM³ without serum and kept at 4°C until processing.

MEDIA

Five Percent (w/v) Trypsin Stock Solution.

Trypsin (Difco 1:250) is added to warm (37°C) Dulbecco’s phosphate buffer saline⁴ without calcium or magnesium (pH 7.7, 330 mOsm) and stirred vigorously at 37°C for 3–4 hours, until the suspension is almost clear. This suspension is then sterilized through a membrane filter (Millipore Corporation, Bedford, Massachusetts) assembly with a prefilter, a 0.8, a 0.45, and a 0.22 µm pore size membranes and sterilized together as a unit. Tryton X-100 free membranes are used and, in addition, the filters are washed with hot (90°C) triple-distilled water immediately before use, to eliminate any other soluble contaminant which may be present.⁵ The sterile trypsin solution can be kept frozen for 3 to 6 months at – 18°C without appreciable loss of activity. The small precipitate which may appear when thawed will usually disappear when the solution is heated to 37°C.

Ten Percent (w/v) Stock Disodium EDTA Solution.

Disodium ethylenediaminetetraacetic acid (ACSS0311 Fisher) is prepared with triple-distilled water. This solution is sterilized by vacuum filtration with an MT-VFA 7–500 Selas Flotronic filter unit with 0.27 μm disposable candles.

Dispersing Solution.

The solution contains 0.25% (w/v) trypsin and 0.02% (w/v) EDTA in Dulbecco’s BSS without calcium or magnesium ions (pH 7.2, 290 mOsm). This solution is prepared immediately before use from the stock solutions.

DISPERSING PROCEDURE

1. Remove and discard the renal capsule and pelvis, and weigh the remaining tissue.

2. Mince the tissue very thoroughly with scissors, using a beaker of appropriate size, until all pieces are approximately 2–3 mm or less.

3. Wash the minced tissue with warm (37°C) Dulbecco’s BSS or CMRL 1415 ATM to eliminate the red blood cells and tissue debris until the supernatant is clear.

4. Wash once with the dispersing solution and place the minced tissue in a trypsinization flask (Bellco Glass, Inc., Vineland, New Jersey) with a Teflon-coated magnetic bar.

5. Add approximately 125 ml of warm (37°C) dispersing solution for each 10 g of tissue. Place the flask on a magnetic stirrer at room temperature and stir as vigorously as possible without producing foam.

6. Stir for intervals (runs) of 15–20 minutes. At the end of each run, stop the stirring for 1 to 2 minutes to allow for the nondispersed fragments to settle, and then decant the supernatant. Repeat these runs until all of the tissue is dispersed. To keep constant the volume relation between the remaining tissue and the added dispersing agent, reduce its amount by approximately 10% for each successive run.

7. After each run, decant and filter the cell suspension through several layers of gauze into a 200-ml round bottom centrifuge bottle. Add an equal volume of growth media with 2 to 10% calf or fetal calf serum to the bottle.

8. Centrifuge the cell suspension immediately for 20 minutes at 90 g at room temperature or at 4°C. Immediately after centrifugation remove the supernatant by suction, and resuspend the cell pellet in growth media by vigorous pipetting.

9. Pool all the cells obtained, as described above, in approximately 100 ml of growth media per each 5–8 g of dispersed tissue, and stir gently with a magnetic stirrer to keep the cells in suspension.

10. Take samples for cell count and cell viability determination. Obtain the percentage of viable cells by using the trypan blue exclusion method.⁶

RESULTS

Simian Kidney.

Results obtained with the method described above, using different dispersing agents, are tabulated in Table I: (1) trypsin 0.25% (w/v); (2) trypsin 0.25% (w/v) disodium EDTA 0.02% (w/v) and, (3) by using the Youngner technique⁷ with trypsin 0.25% (w/v) as dispersing agent.

TABLE I

Cell Yield with Rhesus Monkey Kidneysa

aCourtesy of American Society for Microbiology.

bValues to be multiplied by 10⁶.

The higher percentage of dead cells in the Youngner’s method is due to the centrifugation performed in a conical tube, with all of the dispersed cells, to determine their total volume. When this step is omitted and, instead, cell counts are performed, the number of dead cells is reduced by approximately one-half.

In this and all similar experiments in which different techniques were compared, the minced tissue was distributed in various samples of equal weight and each sample was treated with one of the dispersing agents.

Table II summarizes the results reported in the literature,⁸–¹³ and also presents the results obtained by this author during routine dispersion of 3800 g of simian kidneys. No significant difference was observed between Vervet and Rhesus monkey kidneys. The average cell yield for the first was 133 × 10⁶ cells/g and for the second was 131 × 10⁶ cells/g.

TABLE II

Monkey Kidney Cell Yields Reported by Different Authorsa

aCourtesy of American Society for Microbiology.

bNine grams of tissue per pair of kidneys. Values to be multiplied by 10⁶.

cValues to be multiplied by 10⁶.

dThe flasks were fully covered between 5 and 7 days.

eFifteen grams of tissue per pair of kidneys.

Rabbit Kidney.

An increase in cell yield was obtained with rabbit kidney using trypsin-disodium EDTA, as compared with trypsin alone.

With young rabbits (3 g per pair of kidneys) 72 × 10⁶ cells/g of tissue were obtained by using trypsin alone as a dispersing agent, and 120 × 10⁶ cells/g were obtained with trypsin-disodium EDTA as the dispersing agent following the technique described above. With older rabbits (6 g per pair of kidneys) cell yields of 37 × 10⁶ cells/g and 76 × 10⁶ cells/g were obtained respectively.

Human Kidney.

Kidneys from refrigerated cadavers of newborn babies were procured aseptically no later than 12 hours after death.

Using the above described technique, a cell yield of approximately 10 × 10⁶ cells/g of tissue was obtained.

In experiments presently being conducted, the addition of 0.1% (w/v) of collagenase (Sigma Chemical Co., St. Louis, Missouri) to the trypsin-EDTA dispersing solution, increases the cell yield to 20 × 10⁶ cells/g of tissue. This effect of the collagenase is apparently due to the fact that it dissolves a viscous material which appears during the dissociation of these human kidneys. This viscous material traps inside many cells and thus, when dissociated, the cell yield increases.

References

1. Montes de Oca, H., Probst, P., Grubbs, B. In Vitro. 1966; 2:127. [(Abstr.)].

2. Montes de Oca, H., Probst, P., Grubbs, B. Appl. Microbiol. 1971; 21:90.

3. Healy, G.M., Parker, R.C. J. Cell Biol. 1966; 30:531.

4. Dulbecco, R., Vogt, M. J. Exp. Med. 1954; 99:167.

5. Cahn, R.D. Science. 1967; 155:195.

6. Hoskins, J.M., Meynell, G.G., Sanders, F.K. Exp. Cell. Res. 1956; 11:297.

7. Youngner, J.S. Proc. Soc. Exp. Biol. Med. 1954; 85:202.

8. Cancevici, G., Dima, M., Stoian, I., Crainic, R. Arch. Roum. Pathol. Exp. Microbiol. 1964; 23:239.

9. Dobrova, I.N. Vop. Virusol. 1959; 4:118. [(Transl.)].

10. Wallis, C., Lewis, R.T., Melnick, J.L. Tex. Rep. Biol. Med. 1961; 19:194.

11. Mironova, L.L., Goldrin, N.E., Mamonenko, L.L. Acta Virol. 1963; 7:189.

12. Rappaport, C. Bull. WHO. 1956; 14:147.

13. Kammer, H. Appl. Microbiol. 1969; 17:524.

CHAPTER 2

Trypsin

C. Marine Teleost Fish Tissues

M. Michael Sigel and Annie R. Beasley

Publisher Summary

This chapter discusses the procedure for obtaining dispersed cells from fins of grunts (Haemulon sp.) and snappers (Lutjanus sp.). In the method described in the chapter, it is emphasized that saline and growth medium formulated for other types of tissue culture must be modified for the use with marine teleost fish cells. This modification consists of increasing the final content of NaCl by a constant of 0.06 M. The requirement for extra salt, a reflection of the high osmolality and NaCl content of marine fish sera, is only for marine teleost fish cells and not for the cells of freshwater fishes. Fishes are killed by severing the spinal column immediately posterior to the head, and all of the fins are excised just above the muscled areas. The tissue is soaked in Dakins solution for 3 minutes and then vigorously agitated in M-CMF-PBS. When trypsinization is completed, the harvests are pooled, filtered through four layers of sterile gauze, and centrifuged in the cold at 200 g for 10 minutes. By this procedure, 90–120 ml of suspension containing 106 cells/ml can be obtained from the fins of a single adult fish.

The following procedure is employed for obtaining dispersed cells from fins of grunts (Haemulon sp.) and snappers (Lutjanus sp.). It is essentially an adaptation of the technique described by Clem et al.¹

It should be emphasized that saline and growth medium formulated for other types of tissue culture must be modified for use with marine teleost fish cells. This modification consists of increasing the final content of NaCl by a constant of 0.06 M, as shown in Table I. (This is simply effected by the addition of 1.35 ml of 26% NaCl/100 ml of conventional solution, such as Hanks’ BSS.) The requirement for extra salt, a reflection of the high osmolarity and NaCl content of marine fish sera, is only for marine teleost fish cells and not for cells of freshwater fishes.

TABLE I

Comparative NaCl Concentrations in Standard Reagents and Solutions for Marine Fish Cells

PROCEDURE

Fish are killed by severing the spinal column immediately posterior to the head, and all of the fins are excised just above the muscled areas. (If this is done in the field, the specimens are placed in chilled marine calcium- and magnesium-free phosphate-buffered saline (M-CMF-PBS) containing 10% calf serum plus 400 units penicillin, 200 μg streptomycin, and 2 μg amphotericin B/ml, and kept on ice for transport to the laboratory.) The tissue is soaked in Dakin’s solution² for 3 minutes then vigorously agitated in M-CMF-PBS. It is further sequentially washed for 10-minute periods in 2 additional aliquots of the saline with antibiotics, and all fins from a single fish are transferred to a flask containing 35–50 ml of 0.25% trypsin (1:250 activity) solution in M-CMF-PBS. After incubation at room temperature for 15 minutes, with continuous stirring, the trypsin is discarded and replaced with another aliquot. Twenty minutes later the fluid is harvested and more trypsin is added to the tissue, a procedure which is repeated three times, all at room temperature. As each harvest is made, calf serum is added to it to give a concentration of 10% and the cell suspension is placed in an ice bath. After the final harvest, the tissue should be reduced to bones and a slimy residue of connective tissue.

When trypsinization is completed, the harvests are pooled, filtered through four layers of sterile gauze, and centrifuged in the cold at 200 g for 10 minutes. The supernate is decanted and the cells are resuspended in Marine Basal Medium Eagle, Hanks’ base, supplemented with 10% calf serum plus 10% human serum, and containing 200 units penicillin, 100 µg streptomycin and 1 μg amphotericin B per milliliter. An aliquot of the suspension is used for a cell count and additional growth medium is added to the remainder to give the desired cell concentration.

By this procedure, 90–120 ml of suspension containing 10⁶ cells/ml can be obtained from the fins of a single adult fish.

References

1. Clem, L.W., Moewus, L., Sigel, M.M. Proc. Soc. Exp. Biol. Med. 1961; 108:762.

2. Dakin’s solution: 36 ml concentrated solution (approximately 12%) NaOCl, 1 ml 6 N HCI, 8 g NaHCO3/liter in H2O.

CHAPTER 2

Trypsin

D. Amphibian Tissues¹

Jerome J. Freed

Publisher Summary

This chapter discusses the procedure for preparing kidney cultures from the grass frog, Rana pipiens. In the procedure described in the chapter, frogs are pithed, washed in tap water, and pinned to a dissecting board. The skin is swabbed with 70% alcohol, the abdomen opened and pinned out, and the viscera moved aside. The elongated, purplish mesonephric kidneys lie behind the dorsal peritoneum on either side of the midline. Whitish nodules or masses may be the virus-associated renal adenocarcinoma (Lucke tumor) common in this species. The kidneys are dissected free and transferred to a Petri dish containing wash medium with antibiotics. Kidneys from six frogs are collected, transferred to a dry dish, minced into 1-mm fragments with scissors, and suspended in wash medium. The cultures obtained will gradually become confluent and may be maintained at high cell density by twice-weekly changes of medium.

Primary monolayer cell cultures have been prepared by dissociating adult amphibian tissue with trypsin in a Ca²+- and Mg²+-free balanced salt solution of about 200 mOsmoles osmotic pressure. The following procedure is one of several published for kidney cultures from the grass frog, Rana pipiens.²–⁶

Animals should be healthy; better success is obtained with freshly captured animals than with those stored in artificial hibernation. Frogs are pithed, washed in tap water, and pinned to a dissecting board. The skin is swabbed with 70% alcohol, the abdomen opened and pinned out, and the viscera moved aside. The elongated, purplish mesonephric kidneys lie behind the dorsal peritoneum on either side of the midline. Whitish nodules or masses may be the virus-associated renal adenocarcinoma (Lucke tumor) common in this species.⁷ The kidneys are dissected free and transferred to a Petri dish containing wash medium with antibiotics.⁸

Kidneys from 6 frogs are collected, transferred to a dry dish, minced into 1-mm fragments with scissors, and suspended in wash medium. With a pipette, the mince is transferred to a screw-capped 125-ml Erlenmeyer flask. The fragments are allowed to settle, the fluid removed with a pipette, and fresh wash medium added. When the tissue has been rinsed three times with 20-ml portions of fluid, it is resuspended in a 0.5% solution of trypsin (Difco 1:250) in wash medium, using 3 ml per pair of kidneys. A belted spin-bar is added, and the flask is stirred magnetically for 30 minutes at from 60 to 75 rpm. Two 50-ml centrifuge tubes are placed in crushed ice, and in the neck of each is placed a 3-inch square of sterile gauze, pushed in with a pipette to form a funnel. The tissue in the flask is allowed to settle, the fluid is drawn off with a pipette, and added through the gauze to the centrifuge tube. Fresh trypsin solution is added to the flask (5 ml per pair of kidneys) and stirring continued for 15 minutes. This fluid is added to the cells in the ice bath, and the procedure repeated twice more.

The gauze is removed, the tubes capped, and the cells sedimented by centrifugation for 10 minutes at 500 g. The pellets are each suspended in about 1 ml of wash medium, the suspensions are combined, and the cells counted with a hemocytometer. Red blood cells (ovoid and nucleated) should not be scored. Plastic tissue culture flasks (25 cm²) or 2-ounce prescription bottles are charged with 4–5 ml of growth medium⁸ and inoculated with 2 × 10⁶ tissue cells. After 3 days incubation at 25°C, the medium and unattached cells are removed, and fresh growth medium is added. From 10 to 30% of the inoculated cells will be attached, and they will begin to multiply after about the third day of culture. The cultures will gradually become confluent and may be maintained at high cell density by twice-weekly changes of medium. An agar overlay for detection of virus plaques may be applied.⁹

We have not been successful in subculturing adult anuran kidney cells, although Balls and Ruben¹⁰ have reported success in subculturing adult Xenopus kidney cells, and Gravell¹¹ has isolated a cell line (3 AKRP) from adult Rana pipiens kidney.

References

2. Auclair, W. Nature (London). 1961; 192:467.

3. Shah, V.C. Experientia. 1962; 18:239.

4. Wolff, K., Quimby, M.C. Science. 1964; 144:1578.

5. Freed, J.J., Rosenfeld, S.J. Ann. N. Y. Acad Sci. 1965; 126:99.

6. Malamud, D. Exp. Cell Res. 1967; 45:277.

7. See papers In Biology of Amphibian Tumors (M. Mizell, ed.). Springer-Verlag, 1970 (Special Supplement, Recent Results in Cancer Research).

8. See Section II.

9. Gravell, M., Granoff, A., Darlington, R.W. Virology. 1968; 36:467.

10. Balls, M., Ruben, L.N. Exp. Cell Res. 1966; 43:694.

11. Gravell, M. Virology. 1971; 43:730.

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¹The techniques described were developed with support from Grant AT(11–1)3110 from the Atomic Energy Commission (U.S.A.E.C. Report No. C00-3110-5), U.S.P.H.S. Grants CA-05959, CA-06927, and RR-05539 from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania.

CHAPTER 3

Collagenase Treatment of Chick Heart and Thyroid

S. Robert Hilfer

Publisher Summary

This chapter provides an overview of procedure for collagenase treatment of chick heart and thyroid. Collagenase is available from a number of chemical supply houses as a crude preparation and in purified form from at least one source. Enzymatic dissociation with collagenase and collagenase-containing mixtures has become the method of choice for the preparation of clonal cell cultures because it seems to cause the least damage to cells and allows the greatest plating efficiencies. The procedure discussed in the chapter produces viable single cell suspensions from both heart muscle and thyroid at a wide range of embryonic ages. A higher percentage of the thyroid cells seem to attach after CTC (0.25% collagenase, 0.1% trypsin and 10.0% chicken serum), but the yield and the rapid increase in cell numbers indicate that collagenase alone is better for thyroid cells. The plating efficiencies of the clonal cultures support this conclusion. It should be noted that the difference in cell structure does not seem to be significant for the survival although it might explain the difference in initial attachment.

Enzymatic dissociation with collagenase and collagenase-containing mixtures has become the method of choice for the preparation of clonal cell cultures (i.e., Coon,¹ Spooner,² and Konigsberg³) because it seems to cause the least damage to cells and allows the greatest plating efficiencies. The procedures, furthermore, have been used successfully to separate the cells of many different embryonic and adult tissues by minor adjustments in the basic technique. The method which will be described is based upon the procedure of Cahn et al.⁴ as described in Spooner,² Hilfer and Brown,⁵ and Shain.⁶ It produces viable single cell suspensions from both heart muscle and thyroid at a wide range of embryonic ages.

SOURCE AND PREPARATION

Collagenase is available from a number of chemical supply houses as a crude preparation and in purified form from at least one source (Worthington, Freehold, New Jersey, Cat. No. CLSPA). For most purposes the crude preparations seem to be satisfactory but the added expense of the purified preparation may be justified for particularly delicate cell types. Even the use of the purified preparation does not assure that enzyme activity will be confined to collagen. Although relatively free of proteolytic contamination (Worthington Enzyme Manual, 1972), proteolytic,⁵,⁷ polysaccharidase, and esterase⁸–¹⁰ activities are difficult to remove from the collagenase activity. Partial purification of the crude preparation can be accomplished by chromatography,³ proteolytic activity can be removed with N-ethyl maleimide¹⁰ but so far it has been impossible to remove the esterase and polysaccharidase activities,¹¹ and the polysaccharidase activity can be minimized only by dilution.⁹

The enzyme solution should be prepared in a calcium- and magnesium-free saline (CMF) such as Hanks’ saline and sterilized by pressure filtration through a sterile microcellulose filter (i.e., a GS Millipore filter in a Swinny holder, Millipore Corp., New Bedford, Massachusetts). A sterile concentrate can be stored frozen and diluted as needed. The ability to dissociate seems to be more consistent, however, if a small portion is prepared for immediate use. The final concentration can range from 0.1 to 2.0% in CMF. For embryonic thyroid and heart, 0.25% collagenase in CMF Hanks’ or a mixture of 0.25% collagenase, 0.1% trypsin (such as Bactotrypsin, Difco, Detroit, Michigan) and 10.0% chicken serum (CTC, Coon¹) in CMF Hanks’ saline have both proved to be satisfactory.

DISSOCIATION

The following procedure has been used successfully for 3- to 20-day thyroids and 8- to 16-day heart ventricles from chick embryos. It is recommended that the original method⁴ be read for detailed information on each step of the procedure. The tissue is removed to a 60-mm glass Petri dish containing Hanks’ saline and adherent tissue is dissected away. The ventricles or thyroids are transferred to separate dishes of fresh saline and the larger glands or hearts are minced. Injury can be minimized by using fine, sharp knives and by making the fragments no smaller than 1 mm³. The fragments are transferred to a 25-ml Erlenmeyer flask containing approximately 5 ml CMF Hanks’. Care must be taken not to place too much tissue in each flask or the cell yield will be reduced. The flasks are loosely stoppered (i.e., with an aluminum cap) and incubated 10 minutes in a 5% CO2/95% air atmosphere at 37°C. The CMF Hanks’ is withdrawn and replaced with an equal volume of the dissociation medium. The flasks are stoppered tightly with silicone rubber stoppers and incubated in a shaking water bath at 37°C (80 strokes/minute) for 10 minutes. Adherent mesenchyme can then be stripped from the tissue by either repeated pipetting with a Pasteur pipette or by agitation with a vortex mixer for no more than 10 seconds. The dissociation medium is changed and the flasks incubated under the same conditions. After 15 minutes the flasks are agitated again and the progress of dissociation inspected with a dissecting microscope. The cells which have been released can be withdrawn and held in nutrient medium while the remaining clumps are subjected to additional treatment. It is more practical to leave the suspended cells (which are actually mixed with many partially dissociated cell clumps) in the flask and to incubate for 1 or 2 additional 10-minute periods. Microscopic examination after agitation should be made at the end of each period and, when sufficient separation has occurred, the suspension is transferred to a centrifuge tube. The enzyme action is stopped by filling the tube with cold (4°C) nutrient medium containing serum. The cells are packed by spinning for 3 to 5 minutes at 1000 rpm in a clinical centrifuge. The supernatant is decanted and the cells suspended by pipetting in a small volume of fresh, cold nutrient medium. The larger cell clumps are removed from the suspension by centrifugation at 1000 rpm for 20 to 30 seconds. The suspension is transferred carefully to a fresh tube and brought to a convenient volume for counting. Greater than 98% of the cells should be present as singles. The few remaining doubles and small clusters can be removed by filtration through sterilized nylon cloth (20-µm mesh Nitex; Tobler, Ernst, & Traber, Inc., New York).

VIABILITY

The only real test of the quality of a dissociation procedure is the viability of the cells it produces. Viability can be estimated by a dye exclusion test (i.e., Cahn et al.⁴) but the methods are not necessarily reliable.¹² Inspection by phase microscopy for the absence of irregular outlines and the presence of smooth cell surfaces is a useful procedure. Thyroid cells are usually smoother after collagenase alone and show blebbing and greater size differentials after CTC. Heart cells, on the other hand, tend to be round after CTC and irregular to elongate after collagenase alone. A better estimate of cell damage can be obtained by electron microscopy. Both heart (Fig. 1) and thyroid (Fig. 2) dissociated with CTC show less evidence of internal disruption and surface blebbing than the same tissues separated with collagenase alone (Figs. 3 and 4). Both methods disrupt myofilaments, but the rough endoplasmic reticulum tends to remain intact with CTC.

Figs. 1–4 Electron micrographs of portions of dissociated cells from 12-day embryos. The cells were centrifuged to form a pellet, fixed in glutaraldehyde and postfixed in osmium tetroxide. Sections were stained in aqueous uranyl acetate and lead citrate. Bar represents 1.0 µm. × 8000.

Fig. 1 Heart dissociated with CTC. Surfaces contain fine projections but are relatively smooth. Cytoplasm is vesiculated although a few channels of endoplasmic reticulum are intact. Myofilaments form a disoriented mass (mf). The mitochondrial structure is normal and the nuclei are relatively rounded in outline.

Fig. 2 Heart dissociated with collagenase. The cell surface is uneven and the cytoplasmic membranes are vesiculated, including the Golgi region (g). Myofilaments (mf) are disoriented; mitochondria contain few cristae; and the nuclei tend to be distorted.

Fig. 3 Thyroid dissociated with CTC. The cells have smooth surfaces and the extensive rough endoplasmic reticulum shows little sign of disruption. The interconnected channels contain fine particulate material. The mitochondria and nuclei show no signs of alteration.

Fig. 4 Thyroid dissociated with collagenase. The cell surfaces are blebbed; the rough endoplasmic reticulum shows some vesiculation; and its contents appear to be lost in comparison to Fig. 3 and the gland in vivo. The mitochondria and nuclei appear normal.

The critical test of viability, however, is the ability of the cells to attach and grow in culture. This parameter was assessed by making mass and clonal plates of the cell suspensions in modified Ham’s F12 Medium supplemented with 10% fetal calf serum.² Cells were plated in 5 ml of medium in 60 × 15 Falcon tissue culture dishes (Falcon Plastics, Oxnard, California). Mass cultures were made with 10⁵ cells per dish and clonal cultures with 10² and 10³ cells per dish. The results of typical experiments are summarized in Table I. A higher percentage of the thyroid cells seem to attach after CTC, but the yield (number of cells released per gland) and the rapid increase in cell numbers indicate that collagenase alone is better for thyroid cells. The plating efficiencies of the clonal cultures support this conclusion. It should be noted that the difference in cell structure (cf. Figs. 2 and 4) does not seem to be significant for survival although it might explain the difference in initial attachment. It is interesting that trypsin and EDTA treatment produces a greater cell yield but results in greater cytoplasmic damage and lower percentage attachment.¹³ Heart cells appear to respond better to CTC than to collagenase alone. The cell yield is greater, attachment seems to be better, and in most experiments cell growth is more prolific. Heart cells separated with collagenase alone showed little ability to clone even at 10⁴ cells per dish. It seems clear that both procedures should be tested on any new tissue to be dissociated.

TABLE I

Viability of Cells Dissociated with Collagenase and CTC

aPlating efficiencies as high as 4% for this embryonic age can be obtained by selection of the serum used in the medium (W. G. Sham⁶).

MODE OF ACTION

Since collagenase alone will separate at least some cell types that are not surrounded by large amounts of collagen, it seems unlikely that the basis of cell separation is entirely collagenolytic. The contaminating protease, esterase, and polysaccharidase activities may account for the effect on such cells. The demonstration that it is these contaminants to collagenase that cause the collapse of the branched organization of the salivary⁸,⁹ is in agreement with this suggestion. However, the absence of fibrils near the surfaces of epithelial cells does not exclude the possibility that collagen is present in some other form.

References

1. Coon, H.G. Proc. Nat. Acad. Sci. U. S. 1966; 55:66.

2. Spooner, B.S. J. Cell. Physiol. 1970; 75:33.

3. Konigsberg, I.R. Develop. Biol. 1971; 26:133.

4. Cahn, R.D., Coon, H.G., Cahn, M.B.Wilt F.H., Wessells N.K., eds. Methods in Developmental Biology. Thomas Y. Crowell: New York, 1967; 493.

5. Hilfer, S.R., Brown, J.M. Exp. Cell Res. 1971; 65:246.

6. W. G. Shain Doctoral dissertation, Temple University, Philadelphia, Pennsylvania, 1971.

7. Mitchell, W., Harrington, W. J. Biol. Chem. 1968; 243:4683.

8. Bernfield, M.R., Wessells, N.K. Develop. Biol. Suppl. 1970; 4:195.

9. Bernfield, M.R., Banerjee, S.D., Cohn, R.H. J. Cell Biol. 1972; 52:647.

10. Peterkofsky, B., Diegelmann, R. Biochemistry. 1971; 10:988.

11. M. R. Bernfield personal communication.

12. Levinson, C., Green, J.W. Exp. Cell Res. 1965; 39:309.

13. Hilfer, S.R., Hilfer, E.K. J. Morphol. 1966; 119:217.

CHAPTER 4

Sequential Enzyme Treatment of Mouse Mammary Gland

F.J.A. Prop and G.J. Wiepjes

Publisher Summary

This chapter discusses the method for sequential enzyme treatment of mouse mammary gland. This method first weakens, without completely digesting, the structural constituents of the stroma by collagenase and hyaluronidase, and then in a second step, a general proteolytic enzyme like Pronase or trypsin brings forth the complete dissolution of the stroma and also of cell adhesion, resulting in a monocellular suspension. An important feature of the method, further, is that no means of mechanical disruption is applied during the enzyme treatments. Vigorous repeated pipetting used for the final disruption in many enzymatic methods is the main reason for low viability. The only mechanical step in the method is the initial dissection of the tissue. The cell yield by this method approximates the maximum attainable. If cultured on glass, the 2.5% Pronase-isolated cells show epithelium-like growth; in cultures of 1.25% Pronase-isolated cells, there is admixture of fibroblastlike cells. The method has also been used for the preparation of cell suspensions from mammary tumors.

For obtaining suspensions of viable cells from normal mouse mammary glands, existing methods using trypsin or Pronase or collagenase, etc., were unsatisfactory as to cell yield and/or cell viability. Therefore a method was devised¹ that first weakens—without completely digesting—the structural constituents of the stroma by collagenase and hyaluronidase, and then, in a second step, a general proteolytic enzyme like Pronase or trypsin brings forth the complete dissolution of the stroma and also of cell adhesion, resulting in a monocellular suspension.

An important feature of the method, furthermore, is that no means of mechanical disruption is applied during the enzyme treatments. Vigorous repeated pipetting used for the final disruption in many enzymatic methods is the main reason for low viability. The only mechanical step in the method is the initial dissection of the tissue.

The cell yield by this method approximates the maximum attainable.

PREPARATION OF CELL SUSPENSION

A 9- to 10-week-old virgin CBA mouse is killed by cervical luxation. It is then immersed in 70% ethanol. The skin and the mammary glands are dissected free under the usual aseptic precautions. The mammary glands are taken off and the lymph nodes removed from them. They are placed in a Petri dish which contains a calcium- and magnesium-free balanced salt solution² [CMF: (in g/liter) 8.00 NaCl, 0.30 KCl, 0.05 NaH2PO4·H2O, 0.025 KH2PO4, 1.00 NaHCO3, 2.00 glucose]. The tissue is chopped in CMF using two crossed scalpels fitted with surgical blades (No. 11) using a cutting movement, avoiding as much as possible compression of the tissue. Subsequently, the tissue fragments are incubated for 15 minutes in 15 ml of CMF in a 25-ml Erlenmeyer flask at 36°C on a gyratory shaker (80 rpm, ¾ inch rotation diameter) in order to remove most of the free Ca and Mg ions and to get rid of debris. All through the following steps the gyratory shaker is operated at the same specifications; this means a very gentle movement. The tissue fragments floating at the surface due to the fatty stroma are then transferred by means of a wide-mouthed pipette into 5 ml of a solution containing 0.125% collagenase (Worthington code CLS, activity 200 U/mg) and 0.1% hyaluronidase (Sigma, Type I) dissolved in CMF with 4% demineralized bovine serum albumin (Poviet Producten N. V.). This solution may be kept frozen or prepared fresh and filtered sterile through a 0.45-μm Millipore filter. After incubation on the gyratory shaker for 45 minutes, the much loosened but still adherent tissue is transferred to 5 ml of Medium 199 containing 1.25 or 2.50% Pronase (CalBiochem, B grade). Pronase contains insoluble impurities; sterile filtration is greatly facilitated when these are first removed by spinning at 3000 rpm for 10 minutes. It is extremely important at this stage of the enzyme treatment to avoid any mechanical damage to the cells. One hour incubation on the gyratory shaker effects a complete dissolution of the tissue structure, the result being a suspension of cell clumps and single cells. The procedure is then carried out at room temperature. Twice the volume of serum is added to stop enzyme action and the cells are spun at 1000 rpm for 5 minutes. The supernatant and the floating fat cells are discarded. The pellet is resuspended gently in 20 ml of culture medium (Hanks’ balanced saline, 0.5% lactalbumin hydrolysate, 20% human serum). Should a mucouslike substance prevent resuspension, three drops of a 0.04% DNase solution (Worthington, D) can be added. Resuspension is followed by filtration through a No. 3 sintered-glass filter (pore width 15–40 µm); suction is applied by a water jet pump; filtration should be accomplished before any appreciable vacuum has been built up. The filtrate is a single-cell suspension. The cells are washed twice in culture medium by centrifugation and resuspension. Cell number and viability are checked in a hemocytometer with 0.25% trypan blue in Hanks’ saline. The viability is generally better than 90%. Cell yield from 300 mg wet weight of mammary gland is approximately 5.5 × 10⁶.

APPLICATIONS

If cultured on glass, the 2.5% Pronase-isolated cells show epithelium-like growth (Fig. 1); in cultures of 1.25% Pronase-isolated cells there is admixture of fibroblastlike cells. Starting from this latter material, Visser et al.³ found hormone effects in primary cell cultures of mouse mammary glands.

Fig. 1 + Pronase 2.5%; chiefly epithelial monolayer; 5 days living culture. × 100.

Using Moscona’s technique for making aggregates on the gyratory shaker-the cells obtained by the method described form globular aggregates with an average diameter of 0.2 mm (Fig. 2); in sections these may show histological structures (Fig. 3).

Fig. 2 + Pronase 1.25%; reaggregates, 3 days on the gyratory shaker. × 100.

Fig. 3 Structural development in aggregate; 3 days on the gyratory shaker. H. E. × 100.

The procedure has also been used for the preparation of cell suspensions from mammary tumors. Good yields were obtained from C3H mouse mammary adenocarcinomas (15 × 10⁶ to over 200 × 10⁶ from tumors weighing 500–2000 mg). Figure 4 shows the growth pattern in culture of these cells.

Fig. 4 Monolayer of primary culture from C3H mammary carcinoma. × 100.

Cultures could also be prepared from human breast cancers. For these, enzyme concentrations and incubation times have to be adapted in relation to the structure of the tumor as revealed in a frozen section made immediately after receiving the material from the operating theater. A procedure for preparing cultures from human breast tumors is given by Lasfargues (Section II, Chapter 3).

References

1. Wiepjes, G.J., Prop, F.J.A. Exp. Cell Res. 1970; 61:451–454.

2. Moscona, A. Exp. Cell Res. 1961; 22:455–475.

3. Visser, A.S., de Haas, W.R.E., Kox, C., Prop, F.J.A. Exp. Cell Res. 1972; 73:516.

CHAPTER 5

Nonenzymatic Dissociations

A. Leukocyte Cell Separation on Glass

Y. Rabinowitz*

Publisher Summary

This chapter describes the method for leukocyte cell separation on glass. Viable normal or leukemic leukocytes can be partially or completely separated in glass bead columns on the basis of differences in adherence of the various cell types to glass in the presence or absence of fresh plasma or serum, Mg²+, and Ca²+. For blood collection, Heparin without preservative (phenol) is used as the anticoagulant. For the preparation of leukocyte-rich plasma, sedimentation of the red blood cells (RBC) is hastened by the addition of 6% dextran in saline, 1 ml of dextran per 5 ml of blood. After RBC sedimentation is completed, the supernatant leukocyte-rich plasma is collected by aspiration. The volume of the cell suspension is adjusted, if necessary, to fit onto a suitable stock column (5–75 ml). The white blood cell count is kept under 20,000 per mm³ to avoid overloading the columns. The columns are made from Pyrex glass tubing. The entire column procedure is best carried out in a 37°C room, but good results are obtainable by simply placing the column into an incubator.

Viable normal¹ or leukemic² leukocytes can be partially or completely separated in glass bead columns on the basis of differences in adherence of the various cell types to glass in the presence or absence of fresh plasma or serum, Mg²+, and Ca²+.

BLOOD COLLECTION

Heparin without preservative³ (phenol) is used as the anticoagulant. Panheprin (Abbott No. 6945), 4–5 U.S.P. units per ml of blood, is used for small samples, while 500 ml samples are collected in plastic bags containing 2115 units of heparin (Abbott No. 4686). Addition of 500 extra units of Panheprin to the plastic bags has proved to be desirable to prevent clotting in the columns.

PREPARATION OF LEUKOCYTE-RICH PLASMA

Sedimentation of the red blood cells (RBC) is hastened by the addition of 6% dextran in saline (clinical grade H, average mol. wt. 184,000, Pharmachem Corp., Bethlehem, Pennsylvania)—1 ml of dextran per 5 ml of blood. After RBC sedimentation is completed, the supernatant leukocyte-rich plasma is collected by aspiration. The volume of the cell suspension is adjusted, if necessary, to fit onto a suitable stock column (5–75 ml). The white blood cell count is kept under 20,000 per mm³ to avoid overloading the