CA1206900A

CA1206900A - Hollow glass shell microcarrier for growth of cell cultures, and method of shell manufacture

Info

Publication number: CA1206900A
Application number: CA000417369A
Authority: CA
Inventors: Raymond L. Downs; Robert L. Nolen
Original assignee: KMS Fusion Inc
Current assignee: KMS Fusion Inc
Priority date: 1981-12-21
Filing date: 1982-12-09
Publication date: 1986-07-02
Also published as: GB2151610A; GB2112377A; FR2518569A1; SE8300990L; GB2151610B; SE452892B; GB8501770D0; GB2112377B; SE8300990D0; DE3341772A1

Abstract

HOLLOW GLASS SHELL MICROCARRIER FOR GROWTH
OF CELL CULTURES, AND METHOD OF SHELL MANUFACTURE

Abstract of the Disclosure A hollow silicate glass microsphere for use as a microcarrier in anchorage-dependent cell cultures, and a process for manufacturing such microspheres. The process of manufacture features a method of tailoring the density of the microspheres to the density of the culture medium by first manufacturing the shells over-dense and then immersing the over-dense shells in an etching solution having the density of the culture medium. As the shells become buoyant, they are removed from the solution.

Description

12C~i9(~0 HOLLOW GLASS SHELL MICROCARRIER FOR GROWTH
OF CELL CULTURES, AND MæTHOD OF SHELL MANUFACTURE

The present invention relates to microcarriers for growth of anchorage-dependent cell culturesO More par-ticularly, the invention relates to hollow glass micro-spheres specifically adapted for use as such microcar-riers, and to methods for manufacture of such micro-spheresO A particularly important and yet more specific aspect of the invention relates to methods for adjusting or tailoring the density of hollow glass microspheres for advantageous employment as cell microcarriersO
0 Back round of the rnvention g In the art of growing anchorage-dependent cell tissue ~ultures, it has heretofore been proposed to replace the standard roller bottles and petrie dishes with so-called microcarriers for providing enhanced surface area for cell attachment. The United States patent to Levine et al 4,189,534 proposes, for example,that microcarriers in the form o solid plastic beads be employed. It has been found, however, that plastic microcarriers of th-s type require alteration of electrically charged surface moieties to promote cell attachment, which alteration is difficult to control quantitatively in production and is toxic to some types of cell culture if not properly controlled.

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~Z06~C30 It is also difficult to remove some cell types from the plastic beads. It has also been proposed to employ solid glass beads as cell microcarriers. The art of microcarriers for animal cell cultures in general is reviewed in 3rd General Meeting of ESACT, Oxford 1979, Develop. biol. Standard, _, pp. 109-294 (S. Karger, Basel 1980).
In addition to the foregoing, a significant dis-advantage of microcarriers previously proposed, includ-ing specifically solid beads of plastic or glass, is adifficulty or inability to control or tailor the density of the microcarrier to that of the selected culture mediumO Conventional cell culture media are aqueous in nature and possess densities in the range of 1.03 to 1009 g/cc~ Plastic beads, however, manufactured in accordance with the above-noted Levine et al patent or other techriiques heretofore employed for microcarriers, cannot be controlled to within this density range, let alone to the exact density of a specific mediumO Glass beads typically have a density on the order of 203 g/cc depending upon glass compositionO To avoid settling and compaction of the microcarriers in the growth medium, which tends to inhibit cell growth, it is necessary to stir or otherwise continuously agitate the culture medium.
However, vigorous agitation i5 itself destructive to many cell types~

Objects and Summary of the Invention An object of the present invention is to provide a microcarrier for the culture of anchorage-dependent cell tissues which overcomes some or all of the aforementioned disadvantages of rnicrocarriers as previously proposed.

~2V6900 In particular, it is an objec~ of the present invention to provide a microcarrier of the described type which closely matches the density of a selected culture medium so as to be readily suspendible therein with minimal agitation, and/or which does not require amine salt or other forms of surface treatment for forming potentially toxic surface coupling agents or charged moieties.
Another object of the invention is to provide a microcarrier of the described type from which the cell culture may be readily removed without substantial damage.
In accordance with a first aspect of the present invention, it has been recognized that hollow spherical glass shell~ or microspheres of silicate composition find advantageous employment as microcarriers in anchorage-dependent animal cell cultures~ In particular, it has been found that silicate glass microspheres manufactured using metal organic gel techniques in accordance with the invention to be described do not require electrically charged surface coupling agents, and indeed produce cell quantities in the cultures tested comparable to those produced employing the charged plastic beads previously described. Additionally, the cell cultures may be readily removed from the gla~s shell surfaces using conventional techniques~
The art of manufacturing hollow glass microspheres having a homogeneously integral and essentially isotropic shell wall of finite thic~ness has been developed for other applications. In particular, a number of techniques, including specifically metal organic gel techniques, have heretofore been proposed for manufacturing glass shells to be used as fuel-containers in laser fusion applications~

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These shells generally have a diameter on the order of millimeters or tenths of millimeters and an aspect rati~ -i.eO a ratio of diameter to wall thickness ~ on the order of one hundred. Thiq implies a shell density of on the order to tenths of g/cc for typical silicate glasses, which would be unsuitable for microcarrier applications in aqueous cultures. Insofar as applicants are aware, the art had yet to propose a method for constructing one-piece or isotropic hollow silicate glass microspheres employing metal organic gel techniques and capable of producing shells having a density in excess of 1 g/cc, and specifically in the range of 1.03 to 1.09 g/cc characteristic o conventional cell culture media. ~ence, another object of the invention is to provide such a method and the resulting microsphere product.
In furtherance of the foregoing, another and more specific object of the invention is to provide a method of manufacturing hollow glass micro~pheres having an aspect ratio on the order of 12, as compared with aspect ratios on the order of 100 resulting from metal organic gel techni~ues of the prior artO
Another and related object of the invention is to provide a method of tailoring the density of pre~ormed glass shells.
Briefly stated, in accordance with another important aspect of the invention, the immediately preceding and other objects of the invention are accomplished by initially forming shells having a density in excess of that desired and then surface etching the preformed shells until the desired density is reached. More specifically, the preformed sheils are immersed in an etchant solution lZU6900 having a density equal to the desired shell density and are removed from the solutions as they become buoyant.
As applied specifically to cell microcarriers, the etchant solution may comprise an aqueous solution having a density equal to that in which the microcarriers are to be employed.

Detailed Description of the Invention The state of the art concerning the manufacture of isotropic hollow glass microspheres is exemplified in the 10 United States patents to Veatch et al 3,030,215, Beck et al 3,365,315 and Budrick et al 4,017,290. (The term "isotropic"
is intended to refer to shells formed as a homogeneously integral or one~piece structure, as distinguished for example from shells which comprise two hemishells adhered together.) See also Souers et al "Fabrication of the Glass Microballoon Laser Target," UCRL-51609, September 26, 1974, and 1977 Annual Report of Laser Fusion Research, KMS Fusion, Inc., pages 1-12 to 1-15. Of particular and additional interest relative to manufacture of silicate microspheres from a metal organic gel and gel powder are the United States patents to Budrick et al 4,02~,253 and Downs et al 4,336,338.
In general, the metal organic gel method of glass microsphere manufacture contemplates formation of a gel which includes oxidizable metallic glass-forming com-ponents such as silicon, boron, potassium, sodium, etc.
ard a blowing agent. (The term "silicate glass" as used herein refers to a glass which includes oxides of silicon B with or without other metallic oxides.) The gel is dried and crushed to ~orm gel particles. Generally, and also in ~Z06900 the practice of the present invention, the gel particles may be segregated by size in a sieving operation. Gel particle siæe at this point, which is normally corelated with final shell size and other criteria, is not critical to the present invention which is concerned more with ultimate shell density.
In accordance with known techniques, the crushed and sieved gel particles are then formed into hollow microspheres in a blowing operation as by dropping the same through a tower furnace or oven of the type shown in the above-mentioned copending application or the Budrick '253 patent, for example. The furnace is main-tained at elevated temperature above the gel softening temperature and at which the blowing agent volatilizes to form the shells as the gel particles drop through the furnaceO In accordance with the present invention, however, in order to decrease the aspect ratio of the final shells, the crushed and sieved gel is first sub-jected to an out-gassing operation to drive off some of the blowing agent.
Specifically, a quantity of crushed and sieved gel particles is first placed in an oven and melted to form a foam-like aggregate. The aggregate is then recrushed and resieved in a ~imultaneous operation by placing the aggregate in a stac~ed sieve having a number of ball bearings on each sieve layer. A ~gentle~ recrushing operation of this type is believed to be important to prevent the formation of only useless dust. The re-crushed and resieved particles are then dropped through the tower furnace to form an intermediate shell product.
The melting temperature and time duration of the out-, lZ06900 _7_ gassing operation are determined empirically depending upon the desired final or maximum aspect ratio of the intermediate shell product for any particular glass compo~ition. In the particular example to be described herein, the final desired shell density is in the range of 1.0 to 1.04 g/cc which, for a glass composition density of 203 g/cc, implies an aspect ratio equal to or less than about 12. It was found by trial and error that an out-gassing temperature of 900C and duration of 15 minutes yielded satisfactory results. The recrushed gel particles, which we then placed in the furnace (1500C), were in the size range of 90 to 180 microns. The inter-mediate product shells in this example had a size range of 75 to 250 microns and an aspect ratio o~ 8 to 44.
The intermediate shell product resulting from the blowing operation is then culled to identify those which are to be subjected to the density adjustment or tailor-ing operation. Specifically, the sh~lls are first immersed in a solution which possesses a density at the lower end of the desired range, in this case water at a density of 1.0 g/cc. Floaters, which have a density less than 1.0 g/cc, are discarded. The remainder are then sieve cut to de-sired size, in khis case 10~ to 200 microns, and immersed in a second solution having a den~ity at the upper end of the desired range. In this case, a 5% aqueous solution of sulfuric acid having a density of 1.04 g/cc is appropriate.
The floaters, of course, already possess a density in the desired range and are separated.
The sinker shells in the 1.04 g/cc solution are then subjected to an etching operation in accordance with the :~LZ~36900 invention to reduce the density thereof to lo 04 g/cc.
More specifically, the shells are first immersed in pure carbon tetrachloride ~1.59 g/cc). The sinkers, having a density in excess of 1.59 g/cc are set aside or dis-cardedO The floaters in carbon tetrachloride are then immersed in a solution of 15% sulfuric acid (lolO g/cc) and 4% hydrogen fluoride, the latter being an etching agentO As the shells become buoyant, indicating removal of surface glass and density decline to 1.10 g/cc, they are removed and immersed in a solution of 5% sulfuric acid (1.04 g/cc) and 2% hydrogen ~luoride. Again, shells are removed as they become buoyant, i.e. at a density of 1.04 g/cc. The result is washed in acetone and dried, to form the final product having a size in the range of 81 to 200 microns and a density in the desired range of 1.0 to 1.04 g/cc~
The resulting product has been successfully employed as microcarriers in culturation of the following cells:
human foreskin fibroblast and chick embryo fibroblast in DMEM media with 5% fetal bovine serum, and murine fibro-sarcoma and Walker carcinosarcoma in RPMI media with 10%
fetal calf serumO The microcarrier shells are substantially buoyant in the culture medium and may be readily maintained in suspended state by mild agitation, such as by mild aeration u~ing carbon dio~ide bubbles which are otherwise useful to control medium pH. The glass shell microcarriers may be treated with amine salts for forming surface charge moieties, although this i~ presently believed to be un-necessary. The shells may also be readily coated with a desired material using conventional techniques. of course, the thickness and density of any coating must be taken into consideration during the density tailoring operation.

:~2~:)6900 g In high-volume production of glass shell micro-carriers in accordance with the invention, it is antici-pated and contemplated that the various process steps hereinabove described be fully or at least partially automated. For example, skimming apparatus may be associated with each culling or etching stage for auto-matically removing floaters. Depending upon accuracy of control during the various operations and tolerance of desired final density range, the two-step etching opera-tion herein described by way o~ example may be replacedby one stage, or for that matter increased to three or more stages. Strength of etchant in solution, and there-fore required etchant time, was selected in the example fox best batch control, and may vary depending upon cir-cumstances. Other etchant and/or culling solutions may be employed.
It will be appreciated that final shell density may be more closely controlled than in the exemplar~ 1.0 g/cc to 1.04 g/cc range described herein. For example, if it were desired to produce shells having densities closely clus-tered about 1004 g/cc, the initial culling step in water could be skipped, and the intermeidate shell product could be im~lersed in the 5% aqueous sulphuric acid solution.
Floaters, having a density below 1.04 g/cc would be dis-carded and sinkers would be subjected to the etching operation. In this respect, it will be appreciated that the step of floating in carbon tetrachloride in the example (1.59 g/cc) was for the purpose o~ narxowing the range of densities to be subjected to the etching process, and thereby improving batch quality control. As applied specifically to microcarriers for cell culturation, it has been found that shell density need be controlled only lZ06~00 within a relatively wide 0.04 g/cc range.
It will be further appreciated that the density tailoring aspects of the invention may find advantageous application in other than the field of cell culturationO
See, for example, Wehrenberg et al, I~Shedding PoundS in PlasticS: Microspheres are Moving,~l Mechanical ~ngineer-ng, october 1978, pages 58-63. In this respect, the density of the final shell may vary widely from the exemplary range of loO to 1004 g/cc, and also from the range of 1.03 to lo 09 g/cc for typical cell culture mediaO
Higher densities may be readily obtained by controlling and adjusting the density of the etchant solution to the desired higher density. As mentioned earlier, the para-meters of the gel out-gassing operation (which ~ecreases shell aspect ratio) are determined empirically based upon desired aspect ratio following the blowing operation, which in turn is determined mathematically based upon density of the glass composition employed and desired final shell density and sizeO
The invention claimed is:

Claims

1.
In the combination for cell cultivation comprising a microcarrier adapted for use as growth sites for anchorage-dependent cells and a cell culture medium of predetermined density, the improvement wherein said microcarrier comprises a spherical shell having a homogeneously integral and continuous shell outer surface of silicate glass composition and a density substantially equal to said predetermined density, such that said microcarrier is substantially buoyant in said medium.
2.
The combination set forth in claim 1 wherein said density of said microcarrier is in the range of 1.03 to 1.09 g/cc.

3.
The combination set forth in claim 1 or 2 wherein said shell is hollow and has an aspect ratio of outside diameter to wall thickness no greater than 12.
4.
In a method of growing anchorage-dependent cells using microcarriers in a cell culture medium of predetermined density, the improvement of employing microcarriers comprised of spherical shells having a continuous outer surface of essentially homogeneous silicate glass composition and having an average density substantially equal to said predetermined density, such that said microcarrier is buoyantly suspended in said medium.

5.
In the method of claim 4, the improvement wherein said microcarriers comprise a multiplicity of hollow spherical shells of essentially isotropic silicate glass composition having an average density, determined by shell composition, wall thickness and diameter, substantially equal to said predetermined density, such that said microcarriers exhibit substantially neutral buoyancy in said medium.