- Email: [email protected]
[12] Cloning
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Acridine orange 56 and a fluorescent derivative of disulfonic acid 57 have also been used as a vital stain.
Reagents and Equipment Trypan blue (vital stain, C.I. 23850)-400 mg added to 90 ml water containing 810 mg NaC1, 60 mg K2HPO4, and 50 mg methyl p-hydroxybenzoate. Heat mixture to boiling, cool, and adjust pH to 7.2-7.3 with 1N N a O H (approx. 8 drops). Adjust to final volume of 100 ml. Hemocytometer
Procedure. Prepare cell suspension as described for cell count. In a test tube mix 0.9 ml of cell suspension and 0.1 ml trypan blue. After 5 min, add to hemocytometer. Count blue stained cells as nonviable. Note: Trypan blue has a greater affinity for serum proteins than for cellular protein. 5° If the background shows heavy staining the concentration of dye should be increased or cells should be centrifuged and resuspended prior to staining. Timing of exposure of cells to dye can be critical in that cells continue to take up stain. If this appears to be the case, cells should be removed from the staining solution prior to counting. ~ 56 M. R. Melamed, L. R. Adams, A. Zimring, J. G. Murnick, and K. Mayer, Am. J. Clin. Pathol. 57, 95 (1972). 5~ I. Katz, Tissue Cult. Assoc. Man. 1, 41 (1975).
[12] C l o n i n g By LOLA C. M. REID
A. Introduction A cloned cell population is one derived from a single parental cell. Cloned populations provide the experimenter with the distinct advantage of minimal genetic variability in the model system under study. Methods for isolating single cells and propagating them into a population of cells are called cloning, and the majority of them fall into one of three categories of techniques: 1. Dilution Plating. Suspensions of cells are diluted with a sufficient volume of medium to permit addition of single cells to dishes. 2. Cloning of Anchorage-Dependent Cells. Dilute concentrations of cells are seeded onto one of several possible substrates. The cells are allowed to attach and to grow into colonies of cells. The colonies of cells are then individually subcultured and transferred to other dishes. METHODS IN ENZYMOLOGY,VOL. LVIII
Copyright © 1979by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181958-2
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3. Cloning of Anchorage-Independent Cells. Dilute concentrations of cells are seeded into medium solidified by agar or agarose. Cells capable of growing in suspension in the agar-medium form morulae-shaped colonies. These colonies are transferred individually to culture dishes. All cloning procedures require careful consideration of factors affecting the survival and growth of isolated cells. In the development of cloning techniques it was discovered that cells leak essential nutrients into the medium and secrete chemical messengers that are required for survival and growth. 1-4 In cell culture jargon, the cells are said to "condition the medium," and medium containing these nutrients and chemical messengers is referred to as "conditioned medium." Because an isolated cell in a large volume of medium cannot condition the medium adequately, the ability of the isolated cell to grow in the given medium is a measure of the cell's autonomy to the factors in conditioned medium. A quantitative measure of this is provided by the plating efficiency, the number of colonies of cells (each colony deriving from one cell) that form in plates seeded with dilute concentrations of cells (usually 100-200), calculated as the number of colonies, over the number of cells seeded, multiplied by 100. Many established cell cultures and most primary cell cultures have low plating efficiencies, often less than 1% and as low as 0.001%. Since low plating efficiencies are due to the cells' nutritional needs in low cell densities, special cloning media have been developed that are enriched for amino acids and vitamins. 5-1z The chemical messengers found in conditioned media are provided by feeder layers of cells or by supplementation of the cloning medium with old media from confluent cell populations. The techniques described include several of the most routinely used methods from each of the three categories of cloning techniques, as well as methods for preparing feeder layers and cloning media supplemented with conditioned media. The capillary technique originated by Sanford 13 and used to produce the first clonal cell cultures is effective but tedious i H. Eagle, Science 122, 501 (1955). z H. Eagle, Science 130, 432 (1959). 3 K. Sanford, L. Dupr~e, and A. Covalesky, Exp. Cell Res. 31, 345 (1963). 4 R. G.Ham, Exp. Cell Res. 29, 515 (1963). 5 R. G. Ham, Biochem. Biophys. Res. Commun. 14, 34 (1964). 6 R. G. Ham, Proc. Natl. Acad. Sci. U.S.A. 53, 288 (1965). 7 R. G. Ham, In Vitro 10, 119 (1974). s W. McKeehan, S. Hamilton, and R. G. Ham, Proc. Natl. Acad. Sci. U.S.A. 73, 2023 (1976). W. McKeehan, K. McKeehan, S. Hammon, and R. Ham, In Vitro 13, 399 (1977). 10 S. Iwakata, and J. Grace, N . E State J. Med. 64, 2279 (1964). 11 R. Parker, Spec. Publ., N.Y. Acad. Sci. 5, 503 (1957). 12 j. Morgan, J. Morton, and R. Parker, Proc. Soc. Exp. Biol. Med. 73, 1 (1950). 13 K. K. Sanford, W. R. Earle, and G. D. Likely, J. Natl. Cancer Inst. 9, 229 (194'8).
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even in the simplified form.~4 It has been superceded by easier methods and consequently will not be presented here. Methods for cloning plant cells are essentially analogous to those for mammalian cells (see this volume [41]). Guides to specific nutritional requirements and other modifications necessary for plant cells ls,le (see this volume [41]) and for cells from invertebrates (see this volume [39]) are available. B. Equipment and Supplies Needed for All the Cloning Procedures
Equipment Desk-top centrifuge Inverted phase microscope with a total magnification of x 50 to x 100 Incubator, maintained at 37° with a water-saturated atmosphere containing 5% CO2 in air Biogaard or laminar flow hood. The hood minimizes contamination of cultures. If a hood is not available, one can do reasonably well with an inexpensive, plastic-sheltered area in which an ultraviolet light is placed to minimize pathogens. Hemocytometer for counting cells. Cell counting also can be done with a Coulter counter if it is available. Bunsen burner
Supplies Plastic culture dishes and flasks can be obtained from a number of suppliers. However, the plating efficiency and growth of cells may vary somewhat on the plastics from different commercial suppliers. Therefore, one should select plastics from one company and use them exclusively for all experiments in which data are to be compared. Pipettes: Sterile Pasteur pipettes (7" and 9"). Some pipettes should be plugged with cotton prior to sterilization. Sterile 1- and 5-ml volumetric glass or plastic pipettes Sterile test tubes holding 10 ml Basal media: See Table I for a listing of possible cloning media. Serum: Fetal bovine serum is most commonly used in cloning since it has high concentrations of growth factors and pregnancy hor14K. K. Sanford, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 237-241. Academic Press, New York, 1973. 15 A. C. Hildebrandt, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 244-254. Academic Press, New York, 1973. 16 p. K. Dougall, in"Tissue Culture: Methods and Applications"(P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 261-264. Academic Press, New York, 1973.
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TABLE I CLONING MEDIAa Name Eagle's Minimal Essential Medium (MEM) Ham's Cloning Media F10 F12 F12M F12K MCDB104 RPMI CMRL- 1066 Medium 199
Use
References
Established cell lines
2~
Primary cultures or freshly explanted cells
5-9 4
WI-38 and other fibroblast-llke cells Lymphocytes and leucocytes Embryonic cells Virus-infected cells
6 a 9 20 ~1 n
a The compositions of the media listed can be found in this volume [5] and in the specific references noted. Media should be freshly prepared every 2-3 weeks and stored at 4°. Longer storage results in the loss of labile components such as glutamine. Selection of a medium for a call population to be cloned should be made after assaying the plating efficiency and growth behavior of the cells in the medium. Ham's F12K medium, in particular, has proven to be an excellent cloning medium for many cell types and is used routinely in many laboratories.
mones. However, if cells grow ideally in medium supplemented with another serum type, it should be used preferentially. For some cell lines serum components may be replaced with specific mixtures of hormones and growth factors. These techniques are presented in this volume [6] and have been summarized elsewhere. 17Serum used should be heat-inactivated for 30 min at 56°. Trypan blue solutionm0.1% solution in phosphate buffered saline (PBS) Enzyme solutions--one of the following: Trypsin--0.1% trypsin in PBS (Gibco or Sigma) Collagenase--O. 1% collagenase in PBS (Sigma). There is usually a significant amount of proteolytic activity present in the collagenase purchased from most commercial suppliers. Pronase--0.01% in PBS. A 1% stock solution is prepared and stirred for several hours at 4 ° centrifuged, and the supernatant fluid sterilized by Millipore filtration. The stock solution is diluted to the final concentration prior to use. Particularly effective for fibroblast-like cells; pronase is not as good for epithelial cells. 70% Ethanol solution 27 G. Sato, and L. Reid, in "Biochemistry and Mode of Action of Hormones"(H. V. Rickenberg, ed.), Chapter 7. Academic Press, New York, 1978.
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aASl¢ METHODS
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C. Dilution Plating Techniques In brief, the techniques include the preparation of single cell suspensions from monolayer cultures or from solid tissue pieces in an appropriate cloning media; dilution of the cell suspension to a concentration of 10 cells per milliliter of medium; addition of 0.1 ml of medium to a microtest well; incubation of the cells for several weeks to permit growth of the colonies; and transfer of individual colonies to tissue culture dishes or flasks. The original dilution plating techniques were aimed at adding a single cell to a tissue culture plate or to a test tube. With the realization of the cells' need to adjust the medium with "conditioning factors," the volume of fluid was reduced to approximately 0.1 ml per cell by adding the cell to a small capillary la,14 or to a microdrop under paraffin. TM More recently, the technique has been modified by the introduction of microtest plates (FalconPlastics) containing 96 wells, each well holding up to 0.4 ml. One can add a single cell in 0.1 ml of medium per well and thereby provide the individual cell with its own culture dish and with a sufficiently small amount of fluid to minimize the conditioning factor problem.19.~°
Supplies Necessary for Dilution Plating Techniques Falcon Microtest Plates Tissue culture plates (35 mm diameter) Cloning medium supplemented with 10% serum (or, where feasible, specific hormones and growth factors) Enzyme solution (trypsin is adequate for most cell types)
Procedures 1. The cell cultures to be cloned should be recently subcultured, be in an active state of growth, and show no signs of ill health. 2. Cloning of freshly explanted normal or neoplastic tissue is difficult and almost always requires use of feeder layers or conditioned media (techniques presented in subsequent sections). Tissue or tumor to be cloned should be sterilely dissected from the animal. 3. Single cell suspensions are prepared from monolayer cultures as follows. Remove the medium from the culture plates and rinse the plates twice with PBS. Add 1 ml of the trypsin solution to a 60-mm plate (2-3 ml for larger plates or flasks) of cells and incubate the plates at 37° for 5-15 is A. Lwoff,R. Dulbecco, M. Vogt, and M. Lwoff,Virology 1, 128 (1955). 1~j. A. Robb, Science 170, 857 (1970). ~oj. A. Robb, in "Tissue Culture: Methods and Applications"(p. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 270-274. AcademicPress, New York, 1973.
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min. The plates should be watched carefully to ensure that the time of contact with trypsin is minimized. As soon as the majority of cells are rounded and mostly detached from the plates, squirt the ceils with the conditioned medium supplemented with serum. Most serum contains a trypsin inhibitor (chicken serum is an exception). By squirting the cells with the medium, trypsin is inactivated and detachment of the cells from the plates is completed. To completely inactivate trypsin requires a 9:1 dilution of the trypsin solution with serum-supplemented medium. One can also remove trypsin by centrifuging the cells, removing the supernatant fluid, and resuspending the ceils in the cloning medium. Single cell suspensions are prepared from solid tissues, either normal or neoplastic 42 (see this volume [9]), as follows. The dissected tissue is minced finely with scissors sterilized by dipping them in 95% ethanol. Treat the mince with 0.1% collagenase (Sigma) in PBS at a ratio of 10 ml of enzyme solution to 1 ml of mince. Enzymic digestion should take place in a shaker bath at 37°. After 15 min of enzymic treatment, allow the chunks of tissue to settle to the bottom of the test tube, and pipette the supernatant fluid, containing suspended cells, into another test tube. Centrifuge the cells in a desk-top centrifuge at 900 rpm. Resuspend the pellet in cloning medium. The chunks and tissue mince can be repeatedly treated with collagenase solution until they are totally disaggregated. 4. Count the cells by the trypan blue exclusion assay in order to determine the number of viable cells (see this volume [11]!. 5. Dilute the cell suspensions, whether from the monolayer culture or from the solid tissue dispersion, to a concentration of 10 cells/ml. 6. Use 0.1 ml of the medium containing the cells per well. Repeatedly agitate the cell suspension to ensure homogeneous dispersion of the cells throughout the medium. For rapid inoculation of cells into microtiter wells, a Hamilton repeating dispenser (Hamilton Company, Whittier, California, # PB600-1) is useful. 2° 7. Carefully observe each well and score those containing only one cell. 8. Incubate the cultures in 37° incubators with 5% CO2 in air and ~vith 95% relative humidity. 9. During the growth of the colonies, it should be unnecessary to change the medium. However, if colony formation is unusually slow, and a medium change is required, the spent medium can be gently aspirated from the cultures, and fresh medium added. Leave a residue of fluid over the cells while changing medium to prevent desiccation of the cells. 10. When colonies of 500-600 cells have formed, usually in 2-3 weeks, remove the medium from the wells. Rinse twice with phosphate buffered saline. Add 0.1 ml of trypsin solution per well. Allow the cells to
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round, and then add cloning medium with 10% serum to suspend the cells. Transfer the cells to an appropriate tissue culture dish (35-60-mm plate) or flask. D. Cloning of Anchorage-Dependent Cells The techniques in this category are slight variations of the ones described above in "Dilution Plating Techniques." The preparation and dilution of single cell suspensions are the same as described there. The cells are seeded onto 60-ram tissue culture dishes or on 60-ram petri dishes coated with either collagen or fibrin. By seeding only 100-200 cells per plate, one can obtain large colonies of cells that are sufficiently separated to permit isolation of individual colonies. The technique can be used only for cells that require anchorage and spreading on a substrate in order to grow. This includes most normal cells as well as many established cell cultures.
Supplies Necessary for Cloning of Anchorage-dependent Cells Sterilized cloning rings: metal cylinders that are 6 mm in diameter, 12 mm high, and have 1-mm-thick walls Sterilized Dow-Corning stopcock grease: Layer the grease on a glass petri dish and sterilize. The grease may be kept sterile by taping on the lid and keeping the dish in the hood under a UV lamp. Forceps 60,mm tissue culture dishes or, for collagen- or fibrin-coated substrates, 60-ram petri dishes. 35-ram tissue culture dishes
Procedures 1. Cells vary as to the substrates to which they will anchor. Established cells lines will attach to plastic coated with polycations. However, freshly explanted tissues usually prefer either collagen-coated plates or fibrin-coated plates. Preparation of collagen-coated plates is given elsewhere in this volume [21]. Fibrin-coated plates are prepared as follows: Prepare a solution of cloning medium containing 0.12 units of thrombin per 100 ml of medium. Prepare a solution of 250 mg bovine fibrinogen, 800 mg sodium chloride, and 25 nag sodium citrate per liter of glass-distilled water. Sterilize by filtration. Add 1 ml of the fibrinogen solution and 4 ml of the medium containing thrombin to a tissue culture dish and mix rapidly with a sterile Pasteur pipette. A clear gel will form within several minutes. This technique was first introduced by Schindle& 1 who used it to replace 21 R. Schindler, M. Day, and G. A. Fischer, Cancer Res. 19, 47 (1959).
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agar cloning (described in the next section). Schindler suspended the cells in fibrin and permitted them to form colonies shaped like morulae. However, one can also attach the cells to the surface of the fibrin and observe colony formation. 2. Prepare single cell suspensions from monolayer cultures or from tissues by the methods given in "Dilution Plating Techniques." 3. Depending on the plating efficiency of the cells, select an appropriate concentration of them to yield ultimately 1-10 colonies per plate. For example, with a plating efficiency of 5-10%, one should plate 100 cells per plate. 4. Dilute the cell suspensions to an appropriate density so as to add the correct number of cells per 60-mm dish. The 60-mm dishes hold about 5 ml of medium. 5. The plates are incubated at 37° in 5% CO2 in air and with 95% relative humidity. 6. The following day, single cells that are sufficiently isolated to permit easy cloning are marked by encircling them on the plastic with a grease pencil or felt pen. 7. The cells are allowed to grow for several weeks into colonies containing approximately 500-1000 cells. 8. Remove the medium from the plates and rinse them twice with PBS. Leave a residue of the PBS over the cells to prevent their drying. 9. With forceps sterilized by dipping them into 70% ethanol, dip previously sterilized cloning rings into the petri dish containing stopcock grease. Place the grease-coated end down against the plastic so that the ring encircles a colony of cells and so that the greased end forms a seal with the plastic. 10. Add enzyme solution, e.g., 0.1% trypsin, to each of the rings. Usually 1-2 drops of solution will fill the rings. 11. Incubate the cultures for 5-15 min at 37°, observing the cultures periodically with a phase microscope. When the cultures are rounded and ready to detach, use a 9" Pasteur pipette and pipette the solution gently up and down to complete the detachment process. 12. Pipette the solution of suspended cells into a 35-mm tissue culture dish or 35-mm petri dish coated with collagen or fibrin. Add 2 ml of cloning medium per plate. E. Cloning of Cells that Are Anchorage-Independent for Growth Most cells require anchorage to and spreading on a substratc for growth. However, some cells have acquired the ability to grow in suspension cultures. Factors contributing to this cellular capability include viral transformation (SV40, polyoma), infection of cells with mycoplasma, and
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malignant transformation. 22-25 Puck et al. 26 showed that cells which are anchorage-independent for growth can be cloned in a medium solidified by low concentrations of agar. The method has been expanded into a family of techniques for cloning of cells in semisolid media. The version given below is that of Macpherson 27 who developed methods in which a base layer of agar-medium is overlaid with a thin layer of agar-medium containing the cells to be cloned. By layering the cells at the surface of the base layer, they can be observed readily with an inverted phase microscope. Agar contains acidic and sulfated polysaccharides that are inhibitory to most cells but not to many vitally or malignantly transformed cells. The selective inhibition of the agar-medium is due both to the inability of cells to anchor to the agar matrix and to the inhibitory polyanions within the matrix. Growth of cells in agar-medium has become an assay for viral and/or malignant transformation, since growth of the cells in agar is significantly correlated with tumorigenicity of the cells in immunologically suppressed hosts such as the athymic nude mice. 25"2s'29 To reduce the toxicity of the agar, the polyanions were reduced yielding a matrix referred to as agarose. Agarose is used routinely to clone cell types that are anchorage independent but will not grow in agar due to its toxicity. As noted in Section D, one may also use fibrin gels for cloning cells in suspension. However, cells attach to fibrin even though they do not spread on it and so are got strictly anchorage-independent. 3° Normal cells, as well as transformed ones, will form colonies when embedded within a fibrin matrix.
Supplies Necessary for Cloning Anchorage-Independent Cells Agar: prepare a 1.25% agar stock from Difco Bacto-agar. Preparative details are given below. Cloning medium: prepare a double-strength stock solution. Sterilize by filtration. Tryptose phosphate broth: Sterilize by autoclaving. 22 I. Macpherson, and L. Montagnier, Virology 23, 291 (1964). 23 p. H. Black, Virology 28, 760 (1966). 24 I. Macpherson, J. Cell Sci. 1, 145 (1966). 25 V. Freedman, and S. Shin, Cell 3, 355 (1974). ze T. Puck, P. I. Marcus, and S. J. Cieciura, J. Exp. Med. 103, 273 (1956). 27 I. Macpherson, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 276-280. Academic Press, New York, 1973. zs H. P. Klinger, S. Shin, and V. H. Freedman, Cytogenet. Cell Genet. 17, 185 (1976). zg C. D. Stiles, W. Desmond, L. Chuman, G. Sato, and M. Saier, Jr., Cancer Res. 36, 3300 (1976). ao L. Reid, C. Stiles, M. Rindler, and M. Saier, submitted for publication.
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60-mm petri dishes 35-mm tissue culture dishes
Procedures
1. Agar stock. Add 12.5 g of Difco agar to 800 ml of boiling glassdistilled water and heat until the agar dissolves. Make up to 1 liter with water. Sterilize by autoclaving 80-ml portions of the agar solution in screw-capped bottles of 250-ml capacity. Autoclave under low pressure and with the screw caps loosened. Allow the bottled solutions to come to room temperature. Then tighten the bottle caps. Store at room temperature. 2. Melt the agar stock solution by placing the bottle in boiling water until the agar liquefies. Cool for several minutes and place in a water bath at 44° so that the hot water is above the level of the agar. 3. Prepare the complete agar medium by mixing 80 ml of the cloning medium (double strength) with 20 ml tryptose broth and 20 ml serum in the bottle with the agar. Mix by swirling. 4. Pipette 1-2 ml of medium into 60-ram petri dishes or tissue culture dishes and swirl the plate to evenly coat the solution over the bottom of the dish. Allow the base layers to solidify (10-15 min). 5. Prepare a cell suspension as described under "Dilution Plating Techniques." 6. Dilute the cell suspension with cloning medium to a concentration that is 3-fold that of the final, desired concentration. Mix 1 volume of the cell suspension with 2 volumes of the complete agar solution. 7. Spread the cells over the base layer and swirl the plate to assure even spreading. 8. Incubate the cultures at 37° in an atmosphere of 5% CO2 in air and with 95% relative humidity. 9. Allow the cells to form colonies of 500-1000 cells. 10. Colonies are transferred by sucking them from the agar with a Pasteur pipette and transferring to a sterile test tube containing 1 ml of medium. The colony of cells is pipetted to dissociate it from the agar. 11. The cell suspension is transferred to a 35-mm dish to which is added 2 ml of cloning medium. F. Preparation of Cloning Media, Feeder Layers, and Conditioned Media A listing of some of the media used for cloning various cell types is provided in Table I. All of them employ a basal medium enriched in
162
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particular nutrients, especially those, such as pyruvate and nohessential amino acids, that are lost from the cells into the medium.
Feeder Layers The low plating efficiency of some cells, especially freshly explanted cells, prevents or seriously complicates the cloning procedure. As noted, the low plating efficiency is due in part to the loss of essential nutrients from the isolated cells. However, in the special cloning media listed in Table I these nutrients are provided. The other factors, available in highdensity cultures of cells but not in low density cell cultures, are "chemical messengers" secreted by tho ceils and regulating survival and/or growth. The chemical messengers playing roles in colony formation are referred to as Colony Stimulating Activity factors (CSA). To provide such factors, it is necessary to co-culture the cells to be cloned with a feeder layer of the same or another ceil type. Many established cell lines requiting feeder layers under clonal conditions can use feeder layers of the same cell type. Freshly explanted epithelial cells, whether from normal or neoplastic tissue, often do better when cloned over feeder layers of mesenchymal ceils (L. C. M. Reid, unpublished data). Many of the CSA factors have been or are being purified in a number of laboratories. Several of the bettercharacterized CSA are listed in Table II. al-a7 Except for some anaplastic cell lines, supplementation with only the CSA factors is inadequate for colony formation, suggesting that other factors are necessary. Corroborating this interpretation are the findings by Sato and his colleagues that multiple hormones, growth factors, and transfer factors are essential as supplements to basal media to attain growth of many ceil types 17 (see this volume [6]).
Supplies for Preparing Feeder Layers or Conditioned Medium Microtiter wells (Falcon) 35-mm dishes 60-mm dishes Cloning medium 31 N. 3~ G. 33 T. a4 T. a3 G. 38 T. 27 R.
Dulak and H. Temin, J. Cell. Physiol. 81, 161 (1973). Smith and H. Temin, J. Cell. Physiol. 84, 181 (1974). Chen, J. Mealey, Jr., and R. L. Campbell, J. Natl. Cancer Inst. 55, 1275 (1975). Bradley and M. Sumner, Exp. Biol. Med. Sci. 46, 607 (1968). Price, E. McCullough, and J. Till, Blood 42, 341 (1973). Landau and L. Sachs, Proc. Natl. Acad. Sci. U.S.A. 68, 2540 (1971). Stanley and P. Heard, J. Biol. Chem. 252, 4305 (1977).
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BASIC METHODS
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Procedures
1. Feeder layers may be prepared from any cell population desired. Single cell suspensions are obtained from monolayer cultures or from tissues as described for methods for dilution plating. 2. The suspensions are added to whatever type of dish will be used in the cloning procedure. For microtiter dishes, add 50-100 cells per well; for 35-mm dishes, add 104 cells; for 60-mm dishes, add l&-10 e cells per plate. 3. Incubate the cells at 37° with 5% CO2 in aA air atmosphere and with 95% relative humidity. Allow the cells to attach to the plates and grow for 1 or 2 days. 4. Irradiate the plates with 4000-5000 rad (cobalt-60 or cesium-135) to eliminate all mitotic activity. Mitomycin C has been used to mitotically kill the feeder layer cells; it is used less frequently now, since it has been found to adversely affect the cells being cloned. 5. The method of addition of the cells depends on the cloning technique that is used: a . Dilution technique: The single cells are added directly to wells containing confluent feeder layer. b. Anchorage-dependent technique: Cells to be cloned may be added directly to the feeder layer. For those cells that grow best on collagen or fibrin substrates, polymerize the collagen or fibrin over the feeder layer. The cells to be cloned are then added to the surface of the substrate. c. Anchorage-independent technique: As in (b), the agar or agarose matrix is poured as an overlay onto the feeder layer. Cells to be cloned are then added to the surface of the base agar or agarose layer. 6. For conditioned medium, l-2-day-old medium from confluent feeder layer cultures is removed and mixed at a 3:1 ratio with fresh medium. The medium is used as the cloning medium and changed every 4 - 5 days.
[13] C e l l C u l t u r e C h a r a c t e r i z a t i o n : M o n i t o r i n g for C e l l Identification 1
By W A R D D.
PETERSON, JR., WILLIAM
F.
SIMPSON,
and
BHARATI HUKKU
Techniques and cell culture support systems now available enable any bioscience laboratory to successfully propagate animal cells in vitro. While the relative ease with which cell cultures can now be started or Supported in part by National Cancer Institute Contract NO1 CP 3-3333.
METHODS IN ENZYMOLOGY,VOL. LVIII
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-I2-181958-2
BASIC METHODS
[12]
Acridine orange 56 and a fluorescent derivative of disulfonic acid 57 have also been used as a vital stain.
Reagents and Equipment Trypan blue (vital stain, C.I. 23850)-400 mg added to 90 ml water containing 810 mg NaC1, 60 mg K2HPO4, and 50 mg methyl p-hydroxybenzoate. Heat mixture to boiling, cool, and adjust pH to 7.2-7.3 with 1N N a O H (approx. 8 drops). Adjust to final volume of 100 ml. Hemocytometer
Procedure. Prepare cell suspension as described for cell count. In a test tube mix 0.9 ml of cell suspension and 0.1 ml trypan blue. After 5 min, add to hemocytometer. Count blue stained cells as nonviable. Note: Trypan blue has a greater affinity for serum proteins than for cellular protein. 5° If the background shows heavy staining the concentration of dye should be increased or cells should be centrifuged and resuspended prior to staining. Timing of exposure of cells to dye can be critical in that cells continue to take up stain. If this appears to be the case, cells should be removed from the staining solution prior to counting. ~ 56 M. R. Melamed, L. R. Adams, A. Zimring, J. G. Murnick, and K. Mayer, Am. J. Clin. Pathol. 57, 95 (1972). 5~ I. Katz, Tissue Cult. Assoc. Man. 1, 41 (1975).
[12] C l o n i n g By LOLA C. M. REID
A. Introduction A cloned cell population is one derived from a single parental cell. Cloned populations provide the experimenter with the distinct advantage of minimal genetic variability in the model system under study. Methods for isolating single cells and propagating them into a population of cells are called cloning, and the majority of them fall into one of three categories of techniques: 1. Dilution Plating. Suspensions of cells are diluted with a sufficient volume of medium to permit addition of single cells to dishes. 2. Cloning of Anchorage-Dependent Cells. Dilute concentrations of cells are seeded onto one of several possible substrates. The cells are allowed to attach and to grow into colonies of cells. The colonies of cells are then individually subcultured and transferred to other dishes. METHODS IN ENZYMOLOGY,VOL. LVIII
Copyright © 1979by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181958-2
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3. Cloning of Anchorage-Independent Cells. Dilute concentrations of cells are seeded into medium solidified by agar or agarose. Cells capable of growing in suspension in the agar-medium form morulae-shaped colonies. These colonies are transferred individually to culture dishes. All cloning procedures require careful consideration of factors affecting the survival and growth of isolated cells. In the development of cloning techniques it was discovered that cells leak essential nutrients into the medium and secrete chemical messengers that are required for survival and growth. 1-4 In cell culture jargon, the cells are said to "condition the medium," and medium containing these nutrients and chemical messengers is referred to as "conditioned medium." Because an isolated cell in a large volume of medium cannot condition the medium adequately, the ability of the isolated cell to grow in the given medium is a measure of the cell's autonomy to the factors in conditioned medium. A quantitative measure of this is provided by the plating efficiency, the number of colonies of cells (each colony deriving from one cell) that form in plates seeded with dilute concentrations of cells (usually 100-200), calculated as the number of colonies, over the number of cells seeded, multiplied by 100. Many established cell cultures and most primary cell cultures have low plating efficiencies, often less than 1% and as low as 0.001%. Since low plating efficiencies are due to the cells' nutritional needs in low cell densities, special cloning media have been developed that are enriched for amino acids and vitamins. 5-1z The chemical messengers found in conditioned media are provided by feeder layers of cells or by supplementation of the cloning medium with old media from confluent cell populations. The techniques described include several of the most routinely used methods from each of the three categories of cloning techniques, as well as methods for preparing feeder layers and cloning media supplemented with conditioned media. The capillary technique originated by Sanford 13 and used to produce the first clonal cell cultures is effective but tedious i H. Eagle, Science 122, 501 (1955). z H. Eagle, Science 130, 432 (1959). 3 K. Sanford, L. Dupr~e, and A. Covalesky, Exp. Cell Res. 31, 345 (1963). 4 R. G.Ham, Exp. Cell Res. 29, 515 (1963). 5 R. G. Ham, Biochem. Biophys. Res. Commun. 14, 34 (1964). 6 R. G. Ham, Proc. Natl. Acad. Sci. U.S.A. 53, 288 (1965). 7 R. G. Ham, In Vitro 10, 119 (1974). s W. McKeehan, S. Hamilton, and R. G. Ham, Proc. Natl. Acad. Sci. U.S.A. 73, 2023 (1976). W. McKeehan, K. McKeehan, S. Hammon, and R. Ham, In Vitro 13, 399 (1977). 10 S. Iwakata, and J. Grace, N . E State J. Med. 64, 2279 (1964). 11 R. Parker, Spec. Publ., N.Y. Acad. Sci. 5, 503 (1957). 12 j. Morgan, J. Morton, and R. Parker, Proc. Soc. Exp. Biol. Med. 73, 1 (1950). 13 K. K. Sanford, W. R. Earle, and G. D. Likely, J. Natl. Cancer Inst. 9, 229 (194'8).
i54
BASIC METHODS
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even in the simplified form.~4 It has been superceded by easier methods and consequently will not be presented here. Methods for cloning plant cells are essentially analogous to those for mammalian cells (see this volume [41]). Guides to specific nutritional requirements and other modifications necessary for plant cells ls,le (see this volume [41]) and for cells from invertebrates (see this volume [39]) are available. B. Equipment and Supplies Needed for All the Cloning Procedures
Equipment Desk-top centrifuge Inverted phase microscope with a total magnification of x 50 to x 100 Incubator, maintained at 37° with a water-saturated atmosphere containing 5% CO2 in air Biogaard or laminar flow hood. The hood minimizes contamination of cultures. If a hood is not available, one can do reasonably well with an inexpensive, plastic-sheltered area in which an ultraviolet light is placed to minimize pathogens. Hemocytometer for counting cells. Cell counting also can be done with a Coulter counter if it is available. Bunsen burner
Supplies Plastic culture dishes and flasks can be obtained from a number of suppliers. However, the plating efficiency and growth of cells may vary somewhat on the plastics from different commercial suppliers. Therefore, one should select plastics from one company and use them exclusively for all experiments in which data are to be compared. Pipettes: Sterile Pasteur pipettes (7" and 9"). Some pipettes should be plugged with cotton prior to sterilization. Sterile 1- and 5-ml volumetric glass or plastic pipettes Sterile test tubes holding 10 ml Basal media: See Table I for a listing of possible cloning media. Serum: Fetal bovine serum is most commonly used in cloning since it has high concentrations of growth factors and pregnancy hor14K. K. Sanford, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 237-241. Academic Press, New York, 1973. 15 A. C. Hildebrandt, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 244-254. Academic Press, New York, 1973. 16 p. K. Dougall, in"Tissue Culture: Methods and Applications"(P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 261-264. Academic Press, New York, 1973.
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TABLE I CLONING MEDIAa Name Eagle's Minimal Essential Medium (MEM) Ham's Cloning Media F10 F12 F12M F12K MCDB104 RPMI CMRL- 1066 Medium 199
Use
References
Established cell lines
2~
Primary cultures or freshly explanted cells
5-9 4
WI-38 and other fibroblast-llke cells Lymphocytes and leucocytes Embryonic cells Virus-infected cells
6 a 9 20 ~1 n
a The compositions of the media listed can be found in this volume [5] and in the specific references noted. Media should be freshly prepared every 2-3 weeks and stored at 4°. Longer storage results in the loss of labile components such as glutamine. Selection of a medium for a call population to be cloned should be made after assaying the plating efficiency and growth behavior of the cells in the medium. Ham's F12K medium, in particular, has proven to be an excellent cloning medium for many cell types and is used routinely in many laboratories.
mones. However, if cells grow ideally in medium supplemented with another serum type, it should be used preferentially. For some cell lines serum components may be replaced with specific mixtures of hormones and growth factors. These techniques are presented in this volume [6] and have been summarized elsewhere. 17Serum used should be heat-inactivated for 30 min at 56°. Trypan blue solutionm0.1% solution in phosphate buffered saline (PBS) Enzyme solutions--one of the following: Trypsin--0.1% trypsin in PBS (Gibco or Sigma) Collagenase--O. 1% collagenase in PBS (Sigma). There is usually a significant amount of proteolytic activity present in the collagenase purchased from most commercial suppliers. Pronase--0.01% in PBS. A 1% stock solution is prepared and stirred for several hours at 4 ° centrifuged, and the supernatant fluid sterilized by Millipore filtration. The stock solution is diluted to the final concentration prior to use. Particularly effective for fibroblast-like cells; pronase is not as good for epithelial cells. 70% Ethanol solution 27 G. Sato, and L. Reid, in "Biochemistry and Mode of Action of Hormones"(H. V. Rickenberg, ed.), Chapter 7. Academic Press, New York, 1978.
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aASl¢ METHODS
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C. Dilution Plating Techniques In brief, the techniques include the preparation of single cell suspensions from monolayer cultures or from solid tissue pieces in an appropriate cloning media; dilution of the cell suspension to a concentration of 10 cells per milliliter of medium; addition of 0.1 ml of medium to a microtest well; incubation of the cells for several weeks to permit growth of the colonies; and transfer of individual colonies to tissue culture dishes or flasks. The original dilution plating techniques were aimed at adding a single cell to a tissue culture plate or to a test tube. With the realization of the cells' need to adjust the medium with "conditioning factors," the volume of fluid was reduced to approximately 0.1 ml per cell by adding the cell to a small capillary la,14 or to a microdrop under paraffin. TM More recently, the technique has been modified by the introduction of microtest plates (FalconPlastics) containing 96 wells, each well holding up to 0.4 ml. One can add a single cell in 0.1 ml of medium per well and thereby provide the individual cell with its own culture dish and with a sufficiently small amount of fluid to minimize the conditioning factor problem.19.~°
Supplies Necessary for Dilution Plating Techniques Falcon Microtest Plates Tissue culture plates (35 mm diameter) Cloning medium supplemented with 10% serum (or, where feasible, specific hormones and growth factors) Enzyme solution (trypsin is adequate for most cell types)
Procedures 1. The cell cultures to be cloned should be recently subcultured, be in an active state of growth, and show no signs of ill health. 2. Cloning of freshly explanted normal or neoplastic tissue is difficult and almost always requires use of feeder layers or conditioned media (techniques presented in subsequent sections). Tissue or tumor to be cloned should be sterilely dissected from the animal. 3. Single cell suspensions are prepared from monolayer cultures as follows. Remove the medium from the culture plates and rinse the plates twice with PBS. Add 1 ml of the trypsin solution to a 60-mm plate (2-3 ml for larger plates or flasks) of cells and incubate the plates at 37° for 5-15 is A. Lwoff,R. Dulbecco, M. Vogt, and M. Lwoff,Virology 1, 128 (1955). 1~j. A. Robb, Science 170, 857 (1970). ~oj. A. Robb, in "Tissue Culture: Methods and Applications"(p. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 270-274. AcademicPress, New York, 1973.
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min. The plates should be watched carefully to ensure that the time of contact with trypsin is minimized. As soon as the majority of cells are rounded and mostly detached from the plates, squirt the ceils with the conditioned medium supplemented with serum. Most serum contains a trypsin inhibitor (chicken serum is an exception). By squirting the cells with the medium, trypsin is inactivated and detachment of the cells from the plates is completed. To completely inactivate trypsin requires a 9:1 dilution of the trypsin solution with serum-supplemented medium. One can also remove trypsin by centrifuging the cells, removing the supernatant fluid, and resuspending the ceils in the cloning medium. Single cell suspensions are prepared from solid tissues, either normal or neoplastic 42 (see this volume [9]), as follows. The dissected tissue is minced finely with scissors sterilized by dipping them in 95% ethanol. Treat the mince with 0.1% collagenase (Sigma) in PBS at a ratio of 10 ml of enzyme solution to 1 ml of mince. Enzymic digestion should take place in a shaker bath at 37°. After 15 min of enzymic treatment, allow the chunks of tissue to settle to the bottom of the test tube, and pipette the supernatant fluid, containing suspended cells, into another test tube. Centrifuge the cells in a desk-top centrifuge at 900 rpm. Resuspend the pellet in cloning medium. The chunks and tissue mince can be repeatedly treated with collagenase solution until they are totally disaggregated. 4. Count the cells by the trypan blue exclusion assay in order to determine the number of viable cells (see this volume [11]!. 5. Dilute the cell suspensions, whether from the monolayer culture or from the solid tissue dispersion, to a concentration of 10 cells/ml. 6. Use 0.1 ml of the medium containing the cells per well. Repeatedly agitate the cell suspension to ensure homogeneous dispersion of the cells throughout the medium. For rapid inoculation of cells into microtiter wells, a Hamilton repeating dispenser (Hamilton Company, Whittier, California, # PB600-1) is useful. 2° 7. Carefully observe each well and score those containing only one cell. 8. Incubate the cultures in 37° incubators with 5% CO2 in air and ~vith 95% relative humidity. 9. During the growth of the colonies, it should be unnecessary to change the medium. However, if colony formation is unusually slow, and a medium change is required, the spent medium can be gently aspirated from the cultures, and fresh medium added. Leave a residue of fluid over the cells while changing medium to prevent desiccation of the cells. 10. When colonies of 500-600 cells have formed, usually in 2-3 weeks, remove the medium from the wells. Rinse twice with phosphate buffered saline. Add 0.1 ml of trypsin solution per well. Allow the cells to
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BASIC METHODS
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round, and then add cloning medium with 10% serum to suspend the cells. Transfer the cells to an appropriate tissue culture dish (35-60-mm plate) or flask. D. Cloning of Anchorage-Dependent Cells The techniques in this category are slight variations of the ones described above in "Dilution Plating Techniques." The preparation and dilution of single cell suspensions are the same as described there. The cells are seeded onto 60-ram tissue culture dishes or on 60-ram petri dishes coated with either collagen or fibrin. By seeding only 100-200 cells per plate, one can obtain large colonies of cells that are sufficiently separated to permit isolation of individual colonies. The technique can be used only for cells that require anchorage and spreading on a substrate in order to grow. This includes most normal cells as well as many established cell cultures.
Supplies Necessary for Cloning of Anchorage-dependent Cells Sterilized cloning rings: metal cylinders that are 6 mm in diameter, 12 mm high, and have 1-mm-thick walls Sterilized Dow-Corning stopcock grease: Layer the grease on a glass petri dish and sterilize. The grease may be kept sterile by taping on the lid and keeping the dish in the hood under a UV lamp. Forceps 60,mm tissue culture dishes or, for collagen- or fibrin-coated substrates, 60-ram petri dishes. 35-ram tissue culture dishes
Procedures 1. Cells vary as to the substrates to which they will anchor. Established cells lines will attach to plastic coated with polycations. However, freshly explanted tissues usually prefer either collagen-coated plates or fibrin-coated plates. Preparation of collagen-coated plates is given elsewhere in this volume [21]. Fibrin-coated plates are prepared as follows: Prepare a solution of cloning medium containing 0.12 units of thrombin per 100 ml of medium. Prepare a solution of 250 mg bovine fibrinogen, 800 mg sodium chloride, and 25 nag sodium citrate per liter of glass-distilled water. Sterilize by filtration. Add 1 ml of the fibrinogen solution and 4 ml of the medium containing thrombin to a tissue culture dish and mix rapidly with a sterile Pasteur pipette. A clear gel will form within several minutes. This technique was first introduced by Schindle& 1 who used it to replace 21 R. Schindler, M. Day, and G. A. Fischer, Cancer Res. 19, 47 (1959).
[12]
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agar cloning (described in the next section). Schindler suspended the cells in fibrin and permitted them to form colonies shaped like morulae. However, one can also attach the cells to the surface of the fibrin and observe colony formation. 2. Prepare single cell suspensions from monolayer cultures or from tissues by the methods given in "Dilution Plating Techniques." 3. Depending on the plating efficiency of the cells, select an appropriate concentration of them to yield ultimately 1-10 colonies per plate. For example, with a plating efficiency of 5-10%, one should plate 100 cells per plate. 4. Dilute the cell suspensions to an appropriate density so as to add the correct number of cells per 60-mm dish. The 60-mm dishes hold about 5 ml of medium. 5. The plates are incubated at 37° in 5% CO2 in air and with 95% relative humidity. 6. The following day, single cells that are sufficiently isolated to permit easy cloning are marked by encircling them on the plastic with a grease pencil or felt pen. 7. The cells are allowed to grow for several weeks into colonies containing approximately 500-1000 cells. 8. Remove the medium from the plates and rinse them twice with PBS. Leave a residue of the PBS over the cells to prevent their drying. 9. With forceps sterilized by dipping them into 70% ethanol, dip previously sterilized cloning rings into the petri dish containing stopcock grease. Place the grease-coated end down against the plastic so that the ring encircles a colony of cells and so that the greased end forms a seal with the plastic. 10. Add enzyme solution, e.g., 0.1% trypsin, to each of the rings. Usually 1-2 drops of solution will fill the rings. 11. Incubate the cultures for 5-15 min at 37°, observing the cultures periodically with a phase microscope. When the cultures are rounded and ready to detach, use a 9" Pasteur pipette and pipette the solution gently up and down to complete the detachment process. 12. Pipette the solution of suspended cells into a 35-mm tissue culture dish or 35-mm petri dish coated with collagen or fibrin. Add 2 ml of cloning medium per plate. E. Cloning of Cells that Are Anchorage-Independent for Growth Most cells require anchorage to and spreading on a substratc for growth. However, some cells have acquired the ability to grow in suspension cultures. Factors contributing to this cellular capability include viral transformation (SV40, polyoma), infection of cells with mycoplasma, and
160
BASIC METHODS
[12]
malignant transformation. 22-25 Puck et al. 26 showed that cells which are anchorage-independent for growth can be cloned in a medium solidified by low concentrations of agar. The method has been expanded into a family of techniques for cloning of cells in semisolid media. The version given below is that of Macpherson 27 who developed methods in which a base layer of agar-medium is overlaid with a thin layer of agar-medium containing the cells to be cloned. By layering the cells at the surface of the base layer, they can be observed readily with an inverted phase microscope. Agar contains acidic and sulfated polysaccharides that are inhibitory to most cells but not to many vitally or malignantly transformed cells. The selective inhibition of the agar-medium is due both to the inability of cells to anchor to the agar matrix and to the inhibitory polyanions within the matrix. Growth of cells in agar-medium has become an assay for viral and/or malignant transformation, since growth of the cells in agar is significantly correlated with tumorigenicity of the cells in immunologically suppressed hosts such as the athymic nude mice. 25"2s'29 To reduce the toxicity of the agar, the polyanions were reduced yielding a matrix referred to as agarose. Agarose is used routinely to clone cell types that are anchorage independent but will not grow in agar due to its toxicity. As noted in Section D, one may also use fibrin gels for cloning cells in suspension. However, cells attach to fibrin even though they do not spread on it and so are got strictly anchorage-independent. 3° Normal cells, as well as transformed ones, will form colonies when embedded within a fibrin matrix.
Supplies Necessary for Cloning Anchorage-Independent Cells Agar: prepare a 1.25% agar stock from Difco Bacto-agar. Preparative details are given below. Cloning medium: prepare a double-strength stock solution. Sterilize by filtration. Tryptose phosphate broth: Sterilize by autoclaving. 22 I. Macpherson, and L. Montagnier, Virology 23, 291 (1964). 23 p. H. Black, Virology 28, 760 (1966). 24 I. Macpherson, J. Cell Sci. 1, 145 (1966). 25 V. Freedman, and S. Shin, Cell 3, 355 (1974). ze T. Puck, P. I. Marcus, and S. J. Cieciura, J. Exp. Med. 103, 273 (1956). 27 I. Macpherson, in "Tissue Culture: Methods and Applications" (P. F. Kruse, Jr. and M. K. Patterson, eds.), pp. 276-280. Academic Press, New York, 1973. zs H. P. Klinger, S. Shin, and V. H. Freedman, Cytogenet. Cell Genet. 17, 185 (1976). zg C. D. Stiles, W. Desmond, L. Chuman, G. Sato, and M. Saier, Jr., Cancer Res. 36, 3300 (1976). ao L. Reid, C. Stiles, M. Rindler, and M. Saier, submitted for publication.
[12]
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60-mm petri dishes 35-mm tissue culture dishes
Procedures
1. Agar stock. Add 12.5 g of Difco agar to 800 ml of boiling glassdistilled water and heat until the agar dissolves. Make up to 1 liter with water. Sterilize by autoclaving 80-ml portions of the agar solution in screw-capped bottles of 250-ml capacity. Autoclave under low pressure and with the screw caps loosened. Allow the bottled solutions to come to room temperature. Then tighten the bottle caps. Store at room temperature. 2. Melt the agar stock solution by placing the bottle in boiling water until the agar liquefies. Cool for several minutes and place in a water bath at 44° so that the hot water is above the level of the agar. 3. Prepare the complete agar medium by mixing 80 ml of the cloning medium (double strength) with 20 ml tryptose broth and 20 ml serum in the bottle with the agar. Mix by swirling. 4. Pipette 1-2 ml of medium into 60-ram petri dishes or tissue culture dishes and swirl the plate to evenly coat the solution over the bottom of the dish. Allow the base layers to solidify (10-15 min). 5. Prepare a cell suspension as described under "Dilution Plating Techniques." 6. Dilute the cell suspension with cloning medium to a concentration that is 3-fold that of the final, desired concentration. Mix 1 volume of the cell suspension with 2 volumes of the complete agar solution. 7. Spread the cells over the base layer and swirl the plate to assure even spreading. 8. Incubate the cultures at 37° in an atmosphere of 5% CO2 in air and with 95% relative humidity. 9. Allow the cells to form colonies of 500-1000 cells. 10. Colonies are transferred by sucking them from the agar with a Pasteur pipette and transferring to a sterile test tube containing 1 ml of medium. The colony of cells is pipetted to dissociate it from the agar. 11. The cell suspension is transferred to a 35-mm dish to which is added 2 ml of cloning medium. F. Preparation of Cloning Media, Feeder Layers, and Conditioned Media A listing of some of the media used for cloning various cell types is provided in Table I. All of them employ a basal medium enriched in
162
BASIC METHODS
[12]
particular nutrients, especially those, such as pyruvate and nohessential amino acids, that are lost from the cells into the medium.
Feeder Layers The low plating efficiency of some cells, especially freshly explanted cells, prevents or seriously complicates the cloning procedure. As noted, the low plating efficiency is due in part to the loss of essential nutrients from the isolated cells. However, in the special cloning media listed in Table I these nutrients are provided. The other factors, available in highdensity cultures of cells but not in low density cell cultures, are "chemical messengers" secreted by tho ceils and regulating survival and/or growth. The chemical messengers playing roles in colony formation are referred to as Colony Stimulating Activity factors (CSA). To provide such factors, it is necessary to co-culture the cells to be cloned with a feeder layer of the same or another ceil type. Many established cell lines requiting feeder layers under clonal conditions can use feeder layers of the same cell type. Freshly explanted epithelial cells, whether from normal or neoplastic tissue, often do better when cloned over feeder layers of mesenchymal ceils (L. C. M. Reid, unpublished data). Many of the CSA factors have been or are being purified in a number of laboratories. Several of the bettercharacterized CSA are listed in Table II. al-a7 Except for some anaplastic cell lines, supplementation with only the CSA factors is inadequate for colony formation, suggesting that other factors are necessary. Corroborating this interpretation are the findings by Sato and his colleagues that multiple hormones, growth factors, and transfer factors are essential as supplements to basal media to attain growth of many ceil types 17 (see this volume [6]).
Supplies for Preparing Feeder Layers or Conditioned Medium Microtiter wells (Falcon) 35-mm dishes 60-mm dishes Cloning medium 31 N. 3~ G. 33 T. a4 T. a3 G. 38 T. 27 R.
Dulak and H. Temin, J. Cell. Physiol. 81, 161 (1973). Smith and H. Temin, J. Cell. Physiol. 84, 181 (1974). Chen, J. Mealey, Jr., and R. L. Campbell, J. Natl. Cancer Inst. 55, 1275 (1975). Bradley and M. Sumner, Exp. Biol. Med. Sci. 46, 607 (1968). Price, E. McCullough, and J. Till, Blood 42, 341 (1973). Landau and L. Sachs, Proc. Natl. Acad. Sci. U.S.A. 68, 2540 (1971). Stanley and P. Heard, J. Biol. Chem. 252, 4305 (1977).
[12]
CLONING
163
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164
BASIC METHODS
[13]
Procedures
1. Feeder layers may be prepared from any cell population desired. Single cell suspensions are obtained from monolayer cultures or from tissues as described for methods for dilution plating. 2. The suspensions are added to whatever type of dish will be used in the cloning procedure. For microtiter dishes, add 50-100 cells per well; for 35-mm dishes, add 104 cells; for 60-mm dishes, add l&-10 e cells per plate. 3. Incubate the cells at 37° with 5% CO2 in aA air atmosphere and with 95% relative humidity. Allow the cells to attach to the plates and grow for 1 or 2 days. 4. Irradiate the plates with 4000-5000 rad (cobalt-60 or cesium-135) to eliminate all mitotic activity. Mitomycin C has been used to mitotically kill the feeder layer cells; it is used less frequently now, since it has been found to adversely affect the cells being cloned. 5. The method of addition of the cells depends on the cloning technique that is used: a . Dilution technique: The single cells are added directly to wells containing confluent feeder layer. b. Anchorage-dependent technique: Cells to be cloned may be added directly to the feeder layer. For those cells that grow best on collagen or fibrin substrates, polymerize the collagen or fibrin over the feeder layer. The cells to be cloned are then added to the surface of the substrate. c. Anchorage-independent technique: As in (b), the agar or agarose matrix is poured as an overlay onto the feeder layer. Cells to be cloned are then added to the surface of the base agar or agarose layer. 6. For conditioned medium, l-2-day-old medium from confluent feeder layer cultures is removed and mixed at a 3:1 ratio with fresh medium. The medium is used as the cloning medium and changed every 4 - 5 days.
[13] C e l l C u l t u r e C h a r a c t e r i z a t i o n : M o n i t o r i n g for C e l l Identification 1
By W A R D D.
PETERSON, JR., WILLIAM
F.
SIMPSON,
and
BHARATI HUKKU
Techniques and cell culture support systems now available enable any bioscience laboratory to successfully propagate animal cells in vitro. While the relative ease with which cell cultures can now be started or Supported in part by National Cancer Institute Contract NO1 CP 3-3333.
METHODS IN ENZYMOLOGY,VOL. LVIII
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-I2-181958-2