|Publication number||US2858036 A|
|Publication date||Oct 28, 1958|
|Filing date||Feb 26, 1954|
|Priority date||Feb 26, 1954|
|Publication number||US 2858036 A, US 2858036A, US-A-2858036, US2858036 A, US2858036A|
|Inventors||Wilton R Earle, Highhouse Frederick|
|Original Assignee||Wilton R Earle, Highhouse Frederick|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (10), Referenced by (5), Classifications (6)|
|External Links: USPTO, USPTO Assignment, Espacenet|
Oct. 28, 1958 w. R. EARLE ET AL 2,858,036
CULTURE FLASKS FOR USE WITH PLANE SURFACE SUBSTRATE TISSUE CULTURES Filed Feb. 26, 1954 I I "I 'L INVENTORS 17/. Ta 1?. f/P'FLE,
mas-Ma Man #0056,
ATTORNEY United States Patent CULTURE FLASKS FOR USE WITH PLANE SUR- FACE SUBSTRATE TISSUE CULTURES Wilton R. Earle, Burtonsville, and Frederick Highhouse, Germantown, Md., assignors to the United States of America as represented by the Secretary of Health, Education and Welfare Application February 26, 1954, Serial No. 412,967 4 Claims. (Cl. 215-1) (Granted under Title 35, U. 5. Code (1952), sec. 266) The invention described herein may be manufactured and used by or for the Government of the United States for governmental purposes without the payment to us of any royalty thereon in accordance with the provisions of the act of April 30, 1928 (Ch. 460, 45 Stat. L. 467).
This invention relates to tissue culture flasks particularly adapted for the growth of tissue cells in vitro, and includes a method of producing such flasks.
Procedures have heretofore been published by which tissue cultures can for the first time be set up with high accuracy as replicate cultures, the cells growing on the surface of cellophane or directly on the glass surface of a flask. In this type of culture work, the proliferation of the cells can be followed using special procedures involving the enumeration of the cell nuclei.
These methods have required the provision of new types of culture flasks, since no culture flasks heretofore in use were satisfactory, more particularly, because culture vessels heretofore available were adapted for plasma substrate tissue cultures rather than for cultures growing on surface substrates; it being understood that the term surface substrate cultures denotes cultures in which the cells adhere to and migrate along the surface of a cell support, such as a sheet of cellophane or glass, as distinguished from cultures wherein the cells are embedded in and migrate through a matrix, such as a plasma jell.
Thus the successful development and application of surface substrate methods of tissue culture requires the provision of new and improved culture vessels particularly adapted to maintain uniform conditions for surface substrate proliferation, and to facilitate maintenance and control thereof.
Prior attempts were made to produce surface substrate culture flasks by mold blowing. As glass is blown in a mold, however, the glass stretches unevenly. Therefore, as with all mold blown bottles, the mid part of both the top and bottom walls (the larger walls) of the flask were relatively thick, while at the corners and edges where the top and bottom walls join the sides, the glass was much thinner. Flasks fabricated in this way were, therefore, far from satisfactory since in instances the flask corners were so fragile that the flask would shatter during handling or centrifuging. An even more serious defect was that the thickness of the culture fluid layer over the flask floor was very irregular, being thick along the edges and thin near the middle of the floor. Consequently, there was no assurance that living cells adhering to various areas of the surface of the flask floor were under comparable conditions with respect to the media with which they were in contact.
The present invention has for its principal object the provision of improved tissue culture flasks avoiding the difficulties and curing the disadvantages of the flasks heretofore available. More specific main objects, severally and interdependently, are the provision of a tissue culture flask having an extremely level culture floor, one
having an extremely regular wall thickness; one formed to facilitate observation and low power microscopic examination of the proliferating cells; one adapted for centrifugation; one minimizing the adherence of cells to sloping floors and the exposure of cells above the surface to culture fluid; a small flask proportioned to prevent meniscus effects from interfering excessively with the evenness of distribution of the culture medium on the floor of the flask; and a method of producing such flasks in a new and relatively inexpensive manner.
Other objects and advantages of the invention and special features and procedures contributing to the realization of the main objects will be apparent from the following description of preferred embodiments of the invention.
The invention resides in the novel features of the improved flask construction and in the method of making the same, as hereinafter exemplified, and will be more particularly pointed out in the appended claims.
In the accompanying drawing of illustrated embodiments of the invention:
Figs. 1 and 2 are horizontal and vertical cross sections through one embodiment.
Figs. 3 through 7 are perspective views of the mandrels preferably employed in forming the body of the flask of Figs. 1 and 2.
Figs. 8 and 9 are horizontal and vertical cross sections through a modified embodiment.
Figs. 10 through 13 are perspective views of mandrels preferably employed in forming the body of the flask of Figs. 8 and 9.
Fig. 14 is a diagram indicating how the flasks formed with flat internal roof and floor Walls may have the outer surfaces of these walls shaped into parallelism with their inner surfaces to facilitate low power microscopic inspection and even illumination of the culture.
As shown in the drawings, the present invention is adapted for the production of flasks of various sizes. Small flasks are important for experimental work in which culture areas as small as 15 square centimeters or less are desirable. The larger sizes are needed to afford suflicient culture area for the further proliferation of already established and rapidly growing tissue culture stocks. The optical quality, particularly of the smaller flasks, is important to provide for low-power microscopic examination of the growing tissue cells. This is necessary, since in applicants experience, no quantitative studies can safely ignore the general condition of the cells and the culture as determined by such examination.
Furthermore, as shown in the drawings, the flasks, that preferably have parallel roof and floor walls, in accordance with the invention are laterally tapered at the ends thereof opposite to the throat or neck openings therein, the floor, and preferably also the roof, however, extending in an unbroken plane from the main body portions of the flasks to the extremities of the laterally tapered ends thereof, the advantages of these provisions being more fully explained hereinafter.
Turning now to Figs. 1 and 2, the flask therein shown is typical of one of the larger sizes of flasks currently produced in accordance with the invention, and represents a flask having a floor area of say 60 sq. cm. The floor 1 of the flask in the normal proliferating position thereof is horizontal, as shown in the vertical elevation, Fig. 2, and its area is measured between the side walls 2, 3, and between the end walls 4, 5 and the dam 6, which dam is located between the flask floor l and the flask throat 7 in the shoulder region 8 of the flask. The throat portion or neck 7 of the flask in these larger sizes is usually finished with a beaded edge 9, and is preferably formed of cylindrical tubing of a size scalable with a standard sized stopper or cork of rubber or other suitable .3") material. The roof 10 of the flask that overlies the floor 1 is preferably parallel thereto, and the walls 2, 3, 4, and 5 bounding the floor are preferably vertical.
As best illustrated in Fig. 1, the flask is of generally polygonal form, and as shown in Fig. 2, its floor 1 and roof are interconnected at one end by the shoulder walls 8, and the side wall 2, 3, 4, 5 interconnect the same throughout the remainder of the peripheries of the roof and floor walls. The throat or neck 7 opens through the shoulder walls 8. The side walls 2, 3, 4, 5 define a polygonal area comprising a triangular area between the walls 4 and 5 at the opposite end of the flask from the throat 7, and the floor l is internally planar and extends in a flat unbroken plane from the shoulder walls 8 to the extremity 11 (Pig. 1) of the said triangular area.
By this particular construction, when the flask is upended, free floating cells settle out of the fluid in the extremity 11, or can be separated out into such extremity by slow centrifugation, allowing the supernatant culture fluid to be drawn off and replaced. Furthermore, since the floor l is extended on the same flat level plane clear to the extremity 11, when the cells are washed back into the pool of culture medium M (Fig. 2) or grow along the floor underlying the same, there is no sloping floor near the extremity 11 for the cells to adhere to or proliferate along that might expose such cells above the surface of the culture fluid M, or at least under a thinned area thereof where they would not be subjected to the same nutrient conditions as those in the main body of the flask between the Walls 2 and 3.
Also as shown in Fig. 2, in this embodiment the roof wall 10 is parallel to the floor wall 1, and is also internally planar and extends in a flat unbroken plane from the shoulder walls 8 to the extremity ill of the triangular portions thereof between the walls 4- and 5. This construction is of advantage as it facilitates visual observation or low power microscopic examination of the culture, especially When combined with the features about to be described in connection with Fig. 14, after a brief reference to the presently preferred method of making the flask, that will later be described in greater detail As will be apparent when the method is fully described, the planar internal faces of the floor l and roof 10 are preferably produced by shaping the body of the flask 0n mandrels (Figs. 3-6) with final shaping on a polished, plane faced mandrel (Fig. 7) and thus are level and polished, in the case of the surface of the floor 1, and flat and polished in the case of the under surface of the roof 10. As the glass, in the preferred method of shaping on the mandrel (Fig. 7), is worked to reduce irregularities in its thickness, the outer surfaces of the roof 10 and floor l, in the absence of special provisions, are not optically flat, but may be somewhat irregular, as indicated by the dotted lines 12., 13 in Fig. 14. Thus one of the features of the invention is to provide the roof wall with a flat, planar, external surface 10a parallel to its planar internal flat surface 19b, to thereby provide for, and avoid distortion in, visual observation of the culture, and more particularly low power microscopic observation thereof. The planar surface ltttz preferably extends throughout the entire area of the roof lltl, and may be formed in any suitable way, its formation by grinding and polishing being preferred. Similarly, as shown in Fig. 14, the outer surface lla of the floor 1 may be, at least in part, and preferably throughout all of its extent, formed as a polished surface parallel to the flat planar inner surface of the floor, this being desirable to reduce refractions of light when the cells and culture are illuminated through the floor, and to insure level standing of the interior of the floor when the flask is rested on a plane level surface or the like.
Finally, just as the extension of the floor area as a level plane to the extremity 11 (Fig. 1) insures maximum uniformity of the conditions to which cells are exposed near that end of the flask, so the provision of the dam 6, between the floor area and the throat or neck 7, insures against flowage of the culture medium up into the throat area and presents a steep, rather than a sloping, termination of the floor 1 at that end of the flask. As shown in Fig. 1, the dam 6 is preferably formed in the shoulder area of the flask or at its juncture with the floor area, as will be more fully described in connection with the preferred method of making the flask.
As above mentioned, Figs. 3 through 7 illustrate the types of mandrels employed in making the flasks of which Figs. 1 and 2 are typical. These flasks for purposes of economy are preferably made from round glass tubing such as standard wall Pyrex No. 7740 tubing. In fashioning the body for each flask, a length of tubing is cut that is somewhat longer than the length of the flask body to be made, as will appear later from Table I. The length of tubing is treated in a broad and bushy flame to a temperature at which it is somewhat plastic. While in the flame it is laterally stretched and flattened by rapidly and consecutively passing down into it, and then withdrawing, each of a series of flat and progressively broader mandrels, suitably handled. Three such mandrels, as illustrated in Figs. 3, 4, and 5, are generally used in shaping the tubing for the larger sized flasks to form a blank of rectangular internal cross section throughout its body region. There is then passed into the rectangular cross section blank a mandrel (Fig. 6) of rectangular cross section that has a laterally tapered nose end N the top and bottom faces of which are coplanar with the main faces or roof and floor walls WL of the mandrel. This mandrel is preferably slightly oversize compared to the final mandrel (Fig. 7), as exemplified hereinafter in Table II. The heating being continued, the nose walls 4 and 5 (rough) are formed by flowing the glass of the tube-over the nose end N of the mandrel to form the closed end or apex 11 on the blank. There is then inserted in the so closed blank a precisely dimensioned and highly polished cast iron vacuum mandrel (Fig. 7) having the exact configuration of the interior of the body and closed end of the flask to be made (disregarding shrinkage), and having its broad faces or Walls WP parallel, for shaping and highly polishing the inner surfaces of the roof, sides and floor of the flask. While the blank is being shrunk on the mandrel (the vacuum applied through openings V and the hollow handle R in this operation shrinking all the walls 1, 2, 3, 4, 5, and 10 against the mandrel) the heat is raised somewhat and the preparation is hand worked in the flame to further equalize the thickness of the glass over the top, bottom, sides and pointed end of the mandrel. This final mandrel (Fig. 7) is then withdrawn, the vacuum being relieved, and the formed flask body is cooled.
After a number of form-ed flask bodies have been thus prepared, the part of the so-formed body near its open end is reheated in the flame and the shoulders 8 are then formed by drawing down and shaping, and blowing and working, the extra length of the formed body blank near its open end. The shoulder portion 8 is thus formed with an opening of reduced size to fit the size neck or throat to be applied to the flask. A neck or throat 7, usually of cylindrical tubing (Pyrex in the case mentioned) is then sealed on about the opening, and the fluid restricting ridge or dam 6 near the throat is formed by pressure with a V-shaped tool, after which the end of the throat is headed or fire polished, beading being preferred for the large sized flasks, and in some instances flaring of the throat for better gripping of the stopper being practiced.
The so finished flask is then oven annealed, preferably by raising the annealing oven temperature to 560 C., shutting off the heat, and gradually allowing the oven to cool to room temperature over a period of 12 hours or so.
The flasks as so formed and annealed have sufliciently strong walls to withstand slow speed centrifugation. Be- .pause of the method so far described for forming them,
the outer surfaces of the walls, more particularly of walls 10 and 1, are not truly planar and not necessarily truly parallel to the planar inner surfaces of these walls. To provide for more perfect visual and photographic recording of cell morphology, it is contemplated that at least a part, and preferably all, of the outer side of the roof wall be formed as a planar surface parallel to its internal planar surface, this step preferably being accomplished by grinding and polishing in the same manner normally used in polishing plate glass or optical glass. 10 When desired, similar grinding and polishing may be employed to level the outer surface of the floor wall 1, as well.
As above mentioned, it is desirable to produce these flasks, Whether of small or large floor area, with relatively Wide floor areas, so that even in the case of a small sized flask neither the width nor the length of the body is reduced to a dimension in which the meniscus eifect of a pool of nutrient solution covering the floor of the flask would materially thin the depth of the central portion of the pool. This desideratum also reduces the numbers of sets of mandrels needed to make flasks of a full range of sizes, since several sizes of flasks can be made with one set of mandrels merely by Working on shorter or longer cut sections of glass tubing, so that the change in area for several sizes of flasks is accomplished by change of the length dimension, C minus I, in Figs. 1 and 2.
Turning now to Figs. 8 and 9, it will be noted that the flask therein shown is generally similar to, though smaller than, the flask of Figs. 1 and 2, the principal differences being in the relative size of the throat 7, in the location of the dam 6 at a somewhat more advanced position between the lateral shoulder walls 8, and in the fire polishing of the end 9 of the throat or neck, without heading the same.
In making the smaller series of flasks, typified in Figs. 8 and 9, four mandrels, as illustrated in Figs. 10-13, may be used, instead of the five preferred for making the larger flasks, the method of procedure following that previously set forth.
As typifying the sizes of flasks found particularly desirable for the several stages of tissue culture proliferation, illustrative dimensions of four blanks, referred to in the drawings by reference letters, are set forth in Table I, 45 in which the T number designates the approximate floor area of each tissue flask in square centimeters, the dimensions being given in millimeters, and further details and illustrations pertaining to such flasks are set forth in the inventors article published in the Journal of the National Cancer Institute, volume 14, No. 4, February 1954, pages 841-851, incorporated herein by reference.
Table I.Dimensions of T flasks .in mm. with additional details Flask designation T-9 15 30 Length of initial tube used for making blank.
Outside diameter of original tube used for making his k.
Length of finished blank used for making flask.
A. Overall length 165. E. Outside thickness. 29.5. F. Overall width 72. I. Outside length of bottom angle. 30. D. Outside depth of ridge 4 6. 0.bLength of straight part of 90.
ody. B. Length from ridge to bottom 95.
an e. H. I. D. throat, 15 mm. from tip. 15.5. H. I. D. throat, 3 mm. from tip 16.5. G. O. D. throat, 15 mm. from tip- 13 13 18. End of throat straight. straight. rolled heavy bead rolled bead. Standard Rubber stopper, size 00 00 0 2.
As typifying further the dimensions and other data for mandrels preferably employed for forming these typical flasks, also referred to the drawings by reference letters, illustrative data are set forth in Tables II, III, and IV for three sets of mandrels (dimensions in inches).
Table II.-Mandrels for shaping smaller flasks, e. g. T-9 and T-15 Fig.# 10 11 12 13 Materlal Graphite. Graphite. Graphite. Iron. Finish Ground.. Ground.. Ground" Polished. Nose Angle 60 60.
Radii on corners Dimensions (in inches):
Length JIip S-Straight. TThickness. WWidth K-Nose Length. R-Nose Radius UVao. Port Loca... VVac. Port Dia (Both sides of tool) For location of dimensions see single examples in Figs. 37. Table [IL-Mandrefls for shaping medium sized flasks,
Radii on corners Dimensions (in inches):
For locations of dimensions see single examples in Figs. 37
Table IV.Mandrels for shaping larger flasks, e. g. T-60 Fig. 3 4 5 6 7 Material Graph- Graph- Graph- Graph- Iron ite. ite. ite. ite. Finish Ground. Ground- Ground Ground- Polished. Nose Angle 90. Radii on corners A 12nd .010. .010.
Dimensions (in inches) While there have been described herein what are at present considered preferred embodiments of the invention, it will be obvious to those skilled in the art that many modifications and changes may be made therein without departing from the essence of the invention. It is therefore to be understood that the exemplary embodiments are illustrative and not restrictive of the invention, the scope of which is defined in the appended claims, and that all modifications that come within the meaning and range of equivalency of the claims are intended to be included therein.
1. A tissue culture flask of generally polygonal form comprising roof and floor walls with shoulder walls interconnecting the edges of the same at one end thereof, and side walls interconnecting the same throughout the remainder of the peripheries of the roof and floor walls,
and with a throat opening through said shoulder walls, theside walls defining a polygonal area comprising a triangular, area with, an apex medially thereof at the opposite end'of the flask from said'throat so that when the flask isupended, the free floating cells settle out of the fluid in the apex, or can be separated out into such apex by centrifugation, allowing the supernatant culture fiuid to be drawn off and replaced, said floor wall being internally planar and extending in a flat unbroken plane from said shoulder walls to the apex of said triangular area to insure maximum uniformity of the conditions to which cells are exposed near the triangular end of the flask, said floor wall being terminated in the region of its shoulder walls by an integral upstanding dam separating the floor area of the flask from the throat area thereof.
2. At tissue culture flask according to claim 1, in which the roof wall is parallel to the floor wall, said roof wall also being internally planar and extending in a flat unbroken plane from said shoulder walls to the extremity of said triangular area to facilitate visual observation.
3. A tissue culture flask according to claim 2, said roof wall having a ground and polished external surface paralthe floor when the flask is rested on a plane level surface.
References Cited in the file of this patent UNITED STATES PATENTS Re. 12,604 Bagnall Feb. 12, 1907 D. 20,135 Booth Sept. 9, 1890 D. 39,231 Greig Mar. 31, 1908 1,333,935 OConnor Mar. 16, 1920 1,842,228 Wisner Jan. 19, 1932 1,904,222 Mc C. Halbach Apr. 18, 1933 1,999,525 M-orscholz Apr. 30, 1935 2,378,205 Fevas June 12, 1945 2,486,321 OSullivan Oct. 25, 1949 2,491,848 'Breadner et a1. Dec. 20, 1949
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US1333935 *||Jul 18, 1919||Mar 16, 1920||Reynal O'connor Nicomedes F||Medicine-container|
|US1842228 *||Mar 20, 1930||Jan 19, 1932||Percy Wisner||Liquid container|
|US1904222 *||Mar 9, 1932||Apr 18, 1933||Mcc Halbach Robert||Culture bottle|
|US1999525 *||Jun 2, 1933||Apr 30, 1935||Corning Glass Works||Shaping glass|
|US2378205 *||Sep 29, 1944||Jun 12, 1945||Fevas Anna||Bottle|
|US2486321 *||Oct 14, 1948||Oct 25, 1949||O'sullivan James||Ampoule|
|US2491848 *||Aug 1, 1947||Dec 20, 1949||Gen Electric||Method of manufacturing a glass blank|
|USD20135 *||Jul 31, 1890||Sep 9, 1890||F One||Design for a bottle|
|USD39231 *||Nov 9, 1907||Mar 31, 1908||Design fob a bottle or flask|
|USRE12604 *||Mar 30, 1905||Feb 12, 1907||By Direct||Eeissued feb|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US3945523 *||Apr 14, 1975||Mar 23, 1976||Applied Bioscience||Freeze storage container system|
|US6818438||Sep 30, 2003||Nov 16, 2004||Becton, Dickinson And Company||Culture flask|
|US8329470||Dec 11, 2012||Life Technologies Corporation||Labels, containers, system and method for providing reagents|
|US8652424||Jun 9, 2008||Feb 18, 2014||Life Technologies Corporation||Labels, containers, system and method for providing reagents|
|EP0104001A2 *||Aug 24, 1983||Mar 28, 1984||Hana Biologics, Inc.||Triphasic mycoplasmatales culture device and method and competing microorganism inhibiting device for use therein|
|U.S. Classification||435/304.3, 215/40, 435/288.1|