Abstract:
An improved cell culture medium supplement comprising a sterile solution of Dulbecco&#39;s phosphate buffered saline solution along with amino acids of such constitution and concentration sufficient to supplement growth of the cells to be cultured, and whereby the cell culture medium properties of blood serum are simulated.

Description:
BACKGROUND OF THE INVENTION 
     This application is a continuation-in-part of application Ser. No. 950,748 filed on Oct. 12, 1978 now abandoned. 
    
    
     The present invention relates in general to cell culture media and more particularly to an improved cell culture medium supplement which simulates the cell culture medium properties of blood serum. 
     Various different blod sera have been utilized in the culture of various types of cells. Because of the various problems associated with the use of such sera, one suggested approach has been the partial or total replacement thereof. 
     One such attempt at the partial or total replacement of sera is reported at Vol. XIX, Biotechnology, and Bioengineering (1977), and entitled &#34;Primatone RL in Mammalian Cell Culture Media.&#34; Although these and other efforts have proved satisfactory in some cases and for limited applications, the results were not satisfactory in many other cases. 
     Another approach has been to completely replace the entire cell culture medium rather than to attempt to partially replace sera as a medium supplement. This approach is set forth in U.S. Pat. No. 4,049,494 to Tomei. Tomei&#39;s medium is a clinically defined culture media for the cultivation of BHK-6 cells, which are in turn used to produce a vaccine. As such, Tomei&#39;s medium is a completely chemically defined cultured media which is specific for one particular type of cells only and cannot be utilized as a medium for a wide variety of cells. This feature of Tomei&#39;s medium is particularly disadvantageous. An additional disadvantage of Tomei&#39;s medium is that, because it is a completely defined chemical culture media, its use would require radical change from any media currently in use. Accordingly, a researcher in order to use Tomei&#39;s medium in a continuing specific experiment would have to adapt, if possible, the cell line to this new medium. 
     An additional disadvantage of Tomei&#39;s medium is that it has a pH of 4.3, which is unsuitable for most cell lines, and in general would in fact be cytotoxic. In contrast, standard commercially available media for mammalian cell lines or cell strains (heteroploid-diploid) range in pH from 6.5 to 7.4. Commercially available serum is expected to have a pH of approximately 7.0-7.1. Accordingly, Tomei&#39;s medium would in general be unsuitable as well for use with all standard culture media. 
     A yet further disadvantage of Tomei&#39;s medium is that it contains sodium pyruvate, which is very labile and greatly decreases the shelf life of the medium. Moreover, Tomei&#39;s medium contains phenol red, which in some concentrations, has been shown to have cytotoxic effect to several cell lines. 
     It is accordingly an object of the present invention to provide an improved cell culture medium supplement which materially alleviates the disadvantages of prior art systems. 
     It is an additional object of the present invention to provide an improved cell culture medium supplement which may be used for a wide variety of cell lines and whose use will not necessitate extended adaption of a cell line to the medium as supplemented. 
     It is a further object of the present invention to provide an improved cell culture medium supplement which combines amino acids of such constitution and concentration sufficient to support growth in the cells to be cultured, with an appropriate quantity of Dulbecco&#39;s phosphate buffered saline, whereby the cell culture properties of blood serum are simulated. 
     SUMMARY OF THE INVENTION 
     The improved cell culture medium supplement of the present invention comprises a sterile solution of potassium chloride, potassium phosphate, sodium chloride, monobasic hydrated sodium phosphate, and various amino acids of such composition sufficient to support growth in the cells to be cultured, whereby the beneficial properties of blood serum in that respect are simulated. The amino acids preferably utilized in a preferred embodiment of the present invention are prepared by peptic digest of animal tissue and are available from Humko Sheffield Chemical, Division of Kraft, Inc., and sold under the trademarks Primatone RL, Primatone G, and Primatone HS. 
     In other preferred embodiments of the improved cell culture medium supplement of the present invention one or more of said amino acids may be radioactive, whereby the biological uptake of such amino acid may be monitored in the cells to be cultured. Yet additionally, a lipid may be added, whereby a lipidized serum is simulated. Yet further, calcium chloride and magnesium chloride may be added. Such latter embodiments are useful when splitting a culture of mammalian cells to inactivate the trypsin therein, so that a confluent monolayer of cells may be obtained, without serum addition. 
     The improved cell culture medium supplement of the present invention may be better understood by reference to the examples set forth hereinbelow. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The following examples are illustrative of the composition and method of production of the improved cell culture medium supplement of the present invention. 
     EXAMPLE 1 
     This example illustrates some of the physical properties and the chemical composition by amino acid assay of Primatone RL in milligrams per gram, one of the amino acid compositions which may be preferably utilized in the present invention. 
     
         ______________________________________Physical Properties______________________________________Moisture            3.7%Total Nitrogen      11.6%Amino N/Total N     49.3%Chloride            1.2%pH (2% solution)    7.2Solubility (clear, 30° C.)               120 g/1000 cc______________________________________Amino Acid Assay      Amount                AmountAmino Acid (mg/g)    Amino Acid  (mg/g)______________________________________Lysine     48.0      Alanine     52.1Histidine  20.1      Cystine     Not calculableArginine   26.5      Valine      48.4Aspartic Acid      69.0      Methionine  14.8Threonine  33.0      Isoleucine  24.5Serine     35.6      Leucine     66.8Glutamic Acid      85.1      Tyrosine    9.7Proline    35.7      Phenylalanine                            33.4Glycine    45.9      Tryptophan  7.6______________________________________ 
    
     EXAMPLE 2 
     This example illustrates some of the physical properties and the chemical composition by amino acid assay of Primatone G in milligrams per gram, one of the alternative amino acid compositions which may be preferably utilized in the present invention. 
     
         ______________________________________Physical Properties______________________________________Total Nitrogen  16.45%Amino Nitrogen  2.63%pH, 2% solution 7.0Color           Light Straw ColorClarity         Sparkling ClearChlorides       1.9%Sodium          1.7%Microbiological Content:Standard Plate Count           Less than 10,000/gm.Coliform        Negative on all samples tested.Salmonella      Negative on all samples tested.______________________________________Amino Acid Assay      Amount                AmountAmino Acid (mg/g)    Amino Acid  (mg/g)______________________________________Lysine     38.0      Alanine     84.5Histidine  8.2       Cystine     Not calculableArginine   77.7      Valine      22.1Aspartic Acid      62.1      Methionine  10.1Threonine  18.2      Isoleucine  14.0Serine     37.4      Leucine     29.1Glutamic Acid      97.9      Tyrosine    6.9Proline    122.0     Phenylalanine                            18.8Glycine    194.0     Tryptophan  &lt;0.5______________________________________ 
    
     EXAMPLE 3 
     This example illustrates some of the physical properties and the chemical composition by amino acid assay of Primatone HS in milligrams per gram, one of the further alternative preferred amino acid compositions which may be preferably utilized in the present invention. 
     
         ______________________________________Physical Properties______________________________________Total Nitrogen      12.15%Amino Nitrogen      4.93%Moisture            3.8%pH, 2% solution     7.1Color               Light TanClarity, 2% solution               Sparkling Clear______________________________________Amino Acid Assay      Amount                AmountAmino Acid (mg/g)    Amino Acid  (mg/g)______________________________________Lysine     51.0      Alanine     52.5Histidine  13.6      Cystine     Not calculableArginine   21.9      Valine      36.7Aspartic Acid      65.4      Methionine  15.4Threonine  31.0      Isoleucine  26.2Serine     33.6      Leucine     54.8Glutamic Acid      104.0     Tyrosine    10.2Proline    50.2      Phenylalanine                            25.3Glycine    65.4      Tryptophan  3.3______________________________________ 
    
     EXAMPLE 4 
     Twenty liters of solution for use in the improved cell culture medium supplement of the present invention are made with the following ingredients: 
     Sodium chloride: 160 g/20 liters 
     Potassium chloride: 4 g/20 liters 
     Disodium phosphate Na 2  HPO 4  : 23 g/20 liters 
     Monobasic potassium phosphate KH 2  PO 4  : 4 g/20 liters 
     The above items are weighed out and added in the above order to a 4 liter flask containing approximately 2 liters of deionized, doubled-distilled water. The flask is placed on a magnetic stirrer and stirred to facilitate solution of the above salts without heat. After all the above salts have been dissolved, the solution is brought up to volume with a further quantity of deionized, doubled-distilled water. 
     The pH is checked and then adjusted to 7.0 pH with 1 N HCl. The solution is then filtered through a sterile 293 nm filter pad or through a Gelman cartridge system, and then is aseptically collected into sterile bottles in a sterile hood. After all the solution has been sterilized, quality control samples are checked for sterility, virus and mycoplasma contamination using tests which meet FDA standards, such as nutrient broth, tryptic soy broth, blood agar plates, saubaroud&#39;s agar, and mycoplasma agar, as well as CPE tests on sensitive cell lines, human embryonic lung, kidney tissue, human embryonic foreskin, etc. 
     The above solution is stored at 4° C., but does not need to be refrigerated. Ten kilograms of amino acids are added to a large mixing vat. Approximately, 10 liters of the above salt solution are added to the amino acids while stirring. Then the rest of the above salt solution is added to the vat to bring the mixture to its final volume. 
     In some instances it may be necessary to stir the mixture for about 20 to 30 minutes before total dissolution appears. The solution is then checked for pH and buffering capacity. It is then membrane filtered through a sterile 293 nm stack of membrane filter pads or through a cartridge system using an aseptic fill and a sterile hood. It should be noted that membrane sterilization is not necessary because the solution is autoclavable. However, in certain preferred embodiments membrane filter elements may be used to guarantee that there are no particles greater than 0.22 microns left in the solution. Batch samples are kept and quality control samples are checked for cytotoxicity, growth promotion and sterility. After passing such tests, the solution is ready for use. 
     EXAMPLE 5 
     The above procedure of Example 4 is repeated, except that 1.0 gram per liter of magnesium chloride is added. 
     EXAMPLE 6 
     The procedure of Example 5 is repeated, except the 2 grams per liter of calcium chloride are also added. 
     EXAMPLE 7 
     The procedure of Example 6 is followed. The buffering capacity of the solution is checked and the osmolarity found to be about 300, i.e., within the physiological limits, or is adjusted to 300 milli osmoles. 
     EXAMPLE 8 
     The procedure of Example 4 is followed and utilized in the culture of mammalian cells. Prior to the culture of cells being split, 10% serum is added to inactivate the trypsin present in order to obtain a confluent monolayer of cells. (Other non-serum proteins may be added to inactivate the trypsin: Casein, bovine serum albumin or Lactalbumenhydrolysate). 
     EXAMPLE 9 
     Sodium chloride: 160 g/20 liters 
     Potassium chloride: 4 g/20 liters 
     Disodium phosphate Na 2  HPO 4  : 23 g/20 liters 
     Monobasic potassium phosphate KH 2  PO 4  : 4 g/20 liters 
     The above items are weighed out and added in the above order to a 4-liter Erlenmeyer flask of distilled-deionized water. The salts are dissolved with stirring, without heat to form a first salt solution. 
     Calcium chloride: 20 g/20 liters (anhydrous). 
     Magnesium chloride: 20 g/20 liters (C.6H 2  O). 
     The above ingredients are weighed out and dissolved in 2 liters distilled-deionized water, and then stirred without heat until dissolved to form a second salt solution. The above first and second salt solutions are combined. 
     L-cystine: 0.01 g/20 liters 
     L-tyrosine: 97 g/20 liters 
     The above ingredients are weighed out above and added to 2 liters distilled-deionized water. The mixture is stirred on a magnetic stirrer, adding NaOH (sodium hydroxide) tablets one at a time to facilitate dissolving. When dissolved, the above amino acid solution is added to the combined salt solution. 
     Lysine: 480 g/20 liters 
     Histidine: 201 g/20 liters 
     Arginine: 265 g/20 liters 
     Aspartic Acid: 690 g/20 liters 
     Threonine: 330 g/20 liters 
     Serine: 356 g/20 liters 
     Glutamic Acid: 851 g/20 liters 
     Proline: 357 g/20 liters 
     Glycine: 459 g/20 liters 
     Alanine: 521 g/20 liters 
     Valine: 484 g/20 liters 
     Methionine: 148 g/20 liters 
     Isoleucine: 245 g/20 liters 
     Leucine: 558 g/20 liters 
     Phenylalanine: 334 g/20 liters 
     Tryptophan: 76 g/20 liters 
     The above amino acids are weighed out and added to the above salt and amino acid solution. The volume is brought up to 15 liters with distilled-deionized water, and stirred until dissolved. 
     The volume is brought up to 20 liters with distilled-deionized water. 
     The pH is checked and adjusted to 7.0 with 1 N HCl. The solution is then sterile filtered through a filter pad or cartridge system and is aseptically collected into sterile containers in a sterile hood. 
     Quality control samples are tested in compliance with FDA regulations and for ability to grow and maintain swine testicle (ST) cell lines without addition of serum. 
     The cell culture medium supplement of the present invention is stored at 4° C. until heat stability and sterility are checked. 
     Yet additionally, the calcium chloride and magnesium chloride may be eliminated if calcium ion and magnesium ion concentration are critical for a specific user. 
     The applicant&#39;s cell culture medium supplement of the present invention is being marketed under the trademark SEREX, and the following EXAMPLES 10-16 demonstrate the effectiveness of SEREX in reducing or replacing serum in cell culture media for use with regard to several different types of cells. 
     EXAMPLE 10 
     Primary pekin embryo fibroblast cells are disposed into 35×10 mm six well tissue culture plates (FB-6-TC) supplied by Linbro Chemical Company, Inc. The growth medium utilized was medium 199 with Hank&#39;s salts, sodium bicarbonate, L-glutamine (ISI: Gibco) and gentamicin 100 μg/ml (Scherring). 
     When the growth medium was supplemented with 10% SEREX, the cells grew at a rate comparable to the growth rate produced when 10% fetal bovine serum is used. Using 10% SEREX, confluency did not occur. When 9% SEREX and 1% serum was used, confluency and normal growth occurred. 
     The normal maintenance media was supplemented with 2%-5% fetal bovine serum as a growth media. When 5% SEREX was substituted for the fetal bovine serum, the cells could be maintained in the normal fashion. They could be maintained for up to 10 days without needing growth media. The cells were in good condition using SEREX only. 
     EXAMPLE 11 
     Porcine kidney cell line (PK15) cells were disposed into wells in plastic tissue culture plates having an area of two square centimeters. The Minimum Essential Media (MEM) with antibiotics was added. 
     The following chart sets forth the growth of such porcine kidney cell line cells utilizing SEREX and/or fetal calf serum (FCS): 
     
         ______________________________________% SEREX          1 day after 3 days afteror FCS           seeding     seeding______________________________________10% SEREX only   100% OK     100% OK 5% SEREX only    95% OK     100% OK 4% SEREX only    60% weak   not confluent 5% SEREX-5% FCS 100% OK     100% OK 5% FCS          100% OK     100% OK10% FCS          100% OK     100% OK______________________________________ 
    
     The following chart sets forth the maintenance of such porcine kidney cell line cells using 3 day old monolayers grown to confluency with minimum essential media (MEM) utilizing 10% fetal calf serum or 10% SEREX. 
     
         ______________________________________% SEREX    2 days after changing                      8 days afteror FCS     to maintenance medium                      changing______________________________________5% SEREX only      healthy &amp; confluent                      some floating                      dead cells,                      healthy confluent                      cells5% FCS     same as above   same as above10% SEREX  same as above   same as above______________________________________ 
    
     EXAMPLE 12 
     The following data sets forth the growth of vero cells using varying concentrations of SEREX and/or fetal calf serum (FCS): 
     
         ______________________________________% SEREX     1 day after   3 days afteror FCS      seeding       seeding______________________________________ 5% SEREX only       5-10% granular                     all dead except a       weak looking  few scattered cells9.2% SEREX only       same as above same as above4.2% SEREX  50% granular  95% rough looking,0.83% FCS   vacuolated    granular8.3% SEREX  60% granular  90% granular8.3% FCS    100% OK       100% OK0.9% SEREX10% FCS only       100% OK       100% OK______________________________________ 
    
     The following chart sets forth the maintenance data of vero cells wherein 2 day old monolayers are grown to confluency using M199 and 10% fetal calf serum. The growth medium is discarded and replaced by M199 with SEREX and/or fetal calf serum. 
     
         ______________________________________% SEREX   2 days after changing                     8 days after chang-or FCS    to maintenance medium                     ing to maint. medium______________________________________10% FCS only     healthy &amp; confluent                     floating dead cells                     over thick layers of                     healthy confluent cells 5% FCS only     healthy &amp; confluent                     same as above 9% SEREX healthy &amp; confluent                     50% round dead cells; 1% FCS                   remainder contract-                     ing, moribund10% SEREX all cells thin, 30% round dead cells;  only    contracting     remainder contract-                     ing, moribund______________________________________ 
    
     EXAMPLE 13 
     The following chart sets forth the growth pattern utilizing SEREX and/or fetal calf serum for swine testicle cell line: 
     
         ______________________________________% SEREX         1 day after 3 days afteror FCS          seeding     seeding______________________________________10% FCS         100% OK     100% OK 5% FCS-5% SEREX           100% OK     100% OK 5% SEREX       healthy; fewer                       healthy; 90%           than above  confluent10% SEREX       100% OK     100% OK______________________________________ 
    
     Three day old monolayers of swine testicle cell line were grown with 10% fetal calf serum. The following chart sets forth the maintenance results of partial and/or complete replacement of fetal calf serum with SEREX. 
     
         ______________________________________% SEREX  2 days after changing                      8 days afteror FCS   to maintenance medium                      changing______________________________________10% FCS  healthy &amp; confluent                      healthy % confluent 5% FCS  &#34;                 &#34; 5% FCS- &#34;                 &#34; 5% SEREX 3% FCS  &#34;                 &#34; 5% SEREX    &#34;                 &#34;10% SEREX    &#34;                 &#34;______________________________________ 
    
     EXAMPLE 14 
     The following chart represents the growth pattern of Embryonic Mule Skin (EMS) utilizing Hank&#39;s Medium 199 with SEREX and/or fetal calf serum: 
     
         ______________________________________% SEREX    1 day after     30 days afteror FCS     seeding         seeding______________________________________10% FCS    100% OK         100% OK 5% FCS    healthy, reduced                      100% OK 5% SEREX only      50% dead, growth weak                      95% dead or weak10% SEREX- 100% OK         100% OK 5% FCS______________________________________ 
    
     The following chart sets forth the maintenance pattern obtained using Embryonic Mule Skin cells: 
     
         ______________________________________% SEREX      2 days after changing                        5 days afteror FCS       to maintenance medium                        changing______________________________________10% FCS      100% OK         100% OK 5% FCS-5% SEREX        100% OK         100% OK10% SEREX    100% OK         reduced growth______________________________________ 
    
     EXAMPLE 15 
     The following chart illustrates the growth of Roswell Park Memorial Institute (RPMI) 8226 cell line, a human lymphocyte cell line, utilizing RPMI 1640 as a medium: 
     
         ______________________________________% SEREX      1 day after   3 days afteror FCS       seeding       seeding______________________________________20% FCS      healthy 100% OK                      confluent monolayer10% FCS      &#34;             100% OK 5% FCS-5% SEREX        &#34;             100% OK 4% FCS-6% SEREX        &#34;             100% OK 3% FCS-7% SEREX        100% OK       100% OK 2% FCS-8% SEREX        100% OK       100% OK 1% FCS-9% SEREX        healthy, reduced                      no confluence        number        50% dead10% SEREX only        weak, slow growth                      60% dead, weak______________________________________ 
    
     The following chart illustrates the maintenance patterns obtained for the RPMI cell line using SEREX and/or fetal calf serum: 
     
         ______________________________________% SEREX  2 days after changing                     5 days after changingor FCS   to maintenance medium                     ing to maint. medium______________________________________2% FCS   healthy &amp; confluent                     100% OK5% FCS   &#34;                &#34;1% FCS   healthy, 100% OK some dying, weak1% FCS-  healthy &amp; confluent                     100% OK5% SEREX5% SEREX healthy &amp; confluent                     some dying, weak______________________________________ 
    
     EXAMPLE 16 
     The following chart sets forth the growth of L929 (mouse) cells grown in minimum essential media (MEM) with Earles Salts with L-glutamine. 
     
         ______________________________________% SEREX      1 day after    3 days afteror FCS       seeding        seeding______________________________________10% FCS      healthy 100% OK                       100% OK 9% FCS-1% SEREX        &#34;              &#34; 8% FCS-2% SEREX        &#34;              &#34; 7% FCS-3% SEREX        &#34;              &#34; 6% FCS-4% SEREX        &#34;              &#34; 5% FCS-5% SEREX        &#34;              &#34; 4% FCS-6% SEREX        &#34;              &#34; 3% FCS-7% SEREX        &#34;              &#34; 2% FCS-8% SEREX        weak, contracting                       50% dead 1% FCS-9% SEREX        10% dead, weak 70% dead10% SEREX    some suspended cells                       95% dead, weak______________________________________ 
    
     In regard to maintenance, 3 day old monolayers of L929 cells were cultured using 10% fetal calf serum. The same concentrations used as growth media were used for the maintenance media. As little as 3% fetal calf serum and 7% SEREX were able to maintain the cells for 5-7 days. 
     The above examples are meant to be illustrative of the embodiments of the present invention and no limitation is intended thereby. 
     The percentages of ingredients by dry weight of the composition of Example 4 are, as follows: 
     
         ______________________________________Ingredient         % (by dry weight)______________________________________potassium chloride 0.336monobasic potassium phosphate              0.336sodium chloride    13.4dibasic sodium     1.93phosphateamino acids of such compo-              84.0sition sufficient tosupport growth in thecells to be cultured______________________________________ 
    
     The 84.0% by dry weight of amino acids of such composition sufficient to support growth in the cells to be cultured, where Primatone RL is used as the source of the amino acids, is calculated as follows from the assay of Example 1: 
     L-cystine-trace 
     L-tyrosine 1.26% 
     Lysine 6.25% 
     Histidine 2.62% 
     Arginine 3.45% 
     Aspartic Acid 8.96% 
     Threonine 4.30% 
     Serine 4.75% 
     Glutamic Acid 11.08% 
     Proline 4.65% 
     Glycine 5.98% 
     Alanine 6.78% 
     Valine 6.30% 
     Methionine 1.93% 
     Isoleucine 3.19% 
     Leucine 7.26% 
     Phenylalanine 4.35% 
     Tryptophan 0.99% 
     The 84.0% by dry weight of amino acids of such composition sufficient to support growth in the cells to be cultured, where Primatone G is used as the source of the amino acids, is calculated as follows from the assay of Example 2: 
     L-cystine-trace 
     L-tyrosine 0.69% 
     Lysine 3.79% 
     Histidine 0.89% 
     Arginine 7.76% 
     Aspartic Acid 6.20% 
     Threonine 1.82% 
     Serine 3.73% 
     Glutamic Acid 9.77% 
     Proline 12.18% 
     Glycine 19.37% 
     Alanine 8.43% 
     Valine 2.21% 
     Methionine 1.01% 
     Isoleucine 1.40% 
     Leucine 2.90% 
     Phenylalanine 1.88% 
     Tryptophan 0.05% 
     The 84.0% by dry weight of amino acids of such composition sufficient to support growth in the cells to be cultured, where primatine HS is used as the source of the amino acids, is calculated as follows from the assay of Example 3: 
     L-cystine-trace 
     L-tyrosine 1.30% 
     Lysine 6.45% 
     Histidine 1.73% 
     Arginine 2.79% 
     Aspartic Acid 8.38% 
     Threonine 3.94% 
     Serine 4.27% 
     Glutamic Acid 13.23% 
     Proline 6.38% 
     Glycine 8.38% 
     Alanine 6.68% 
     Valine 4.67% 
     Methionine 1.96% 
     Isoleucine 3.33% 
     Leucine 6.97% 
     Phenylalanine 3.22% 
     Tryptophan 0.42% 
     The percentage of ingredients by dry weight of Example 6 are, as follows: 
     
         ______________________________________Ingredient         % (by dry weight)______________________________________potassium chloride .335monobasic potassium phosphate              .335sodium chloride    13.4dibasic sodium     1.92phosphatecalcium chloride   0.167magnesium chloride 0.167amino acids of such compo-              83.7sition sufficient tosupport growth in thecells to be cultured______________________________________ 
    
     These compositions and methods may be widely modified in the accordance with the principles and purposes of the present invention, one such purpose being to provide an improved cell culture medium supplement which is formulated from readily available ingredients. 
     In alternative preferred embodiments where a gel consistency is needed for collogen experimentation or chromatography, the Primatone G amino acid composition of Example 2 with its greater viscosity may be preferable. For other applications, such as where a slight color variation may be particularly useful as spectrophotometric and other color related experimentation, the Primatone HS of Example 3 is preferable. 
     Other modifications, substitutions and alternatives will be readily apparent to one skilled in the art upon a review of the present specification and are intended to be included within the scope of the invention.