GB2151610A - Hollow glass shell microcarrier for growth of cell cultures, and method of shell manufacture - Google Patents

Abstract

A hollow silicate glass microsphere adapted 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

1 GB 2 151 61 OA 1

SPECIFICATION

Hollow glass shell microcarrier for growth of cell cultures, and method of shell manufacture 1.

The present invention relates to microcarriers for growth of anchorage-dependent cell cul tures. More particularly, the invention relates to hollow glass microspheres specifically. 75 adapted for use as such microcarriers, and to methods for manufacture of such micros pheres. A particularly important and yet more specific aspect of the invention relates to methods for adjusting or.tailoring the density. 80 of hollow glass microspheres for advan tageous, employment as cell microcarriers In the art of growing anchorage-dependent cell tissue' cultures, it has heretofore been proposed to replace the standard roller bottles 85 and petrie dishes with so-called. microcarriers for providing enhanced surface area for cell attachment. The. United States patent to Lev ine et al 4,189,534 proposes, for example, that microcarriers in.the form of solid plastic 90 beads be employed. It has been found, how ever, that plastic microcarriers of this type require alteration of electrically charged sur face -moieties to promote cell attachment, which alteration is difficult to control quantita- 95 tively in production and is toxic to some types of cell culture if not properly controlled. 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, - 100 The art of microcarriers for animal cell cultures in general is reviewed in 3rd General Meeting of ESACT, Oxford 1979, Develop. biol. Standard, 46, pp. 109-294 (S. Karger, Basel 1980).

In addition to the foregoing, a significant disadvantage of microcarriers previously proposed, including specifically solid beads of plastic or glass, is a difficulty or inability to control or tailor the density of the microcarrier to that of the selected culture medium. Con ventional cell culture media are aqueous in nature and posse ' ss densities in the range of 1.03 to 1.09 g/cc. Plastic beads, however, manufactured in accordance with the above- 115 noted Levine et al patent or other techniques heretofore employed for microcarriers, cannot be controlled to within this density range, let alone to the exact density of a specific me- dium. Glass beads typically have a density on the order of 2.3 g/cc depending upon glass composition, 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 is itself destructive to many cell types.

An object of the present inventiop is to provide a microcarrier for the culture of anchorage-dependent cell tissues which overcomes some or all of the aforementioned disadvantages of microcarriers as previously proposed. In particular, it is an object 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 shells 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 glass shell surface using conventional techniques.

- The art of manufacturing hollow glass mi crospheres having a homogeneously integral and essentially isotropic shell wall of finite thickness 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 fuelcontainers in laser fusion applications. These shells generally have a diameter on the order of millimeters or tenths of millimeters and an aspect ratio-i.e. a ratio of diameter to wall thickness---on the order of one hundred. This 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 of conventional cell culture media. Hence, 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 microspheres having an aspect ratio on 2 GB 2 151 61 OA 2 the order of 12, as compared with aspect ratios on the order of 100 resulting from metal organic gel techniques of the prior art.

Another and related object of the invention is to provide a method of tailoring the density of preformed glass shells.

Briefly stated, in accordance with another important aspect of the invention, the immediately preceding and other objects of the inven- tion 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 shells are immersed in an etchant solution having a density equal to the desired shell density and are removed from the solution 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.

The state of the art concerning the manufacture of isotropic hollow glass microspheres is exemplified in the 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-51 609, September 26, 19 74, and 19 7 7 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 gfel powder are the United States patent to Budrick et al 4,021,253 and copending United States Application Serial No. 178,266 filed August 15, 1980.

In general, the metal organic gel method of glass microsphere manufacture contemplates formation of a gel which includes oxidizable metallic glass-forming components such as silicon, boron, potassium, sodium, etc. and a blowing agent. (The term -silicate glass- as used herein refers to a glass which includes oxides of silicon with or without other metallic oxides.) The gel is dried and crushed to form gel particles. Generally, and also in the practice of the present invention, the gel particles may be segregated by size in a sieving operation. Gel particle size at this point, which is normally correlated with final shell size and other criteria, is not critical to the present invention which is concerned more with ulti- mate 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 maintained 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 furnace. 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 subjected 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 simultaneous operation by placing the aggregate in a stacked 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 recrushed 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-gassing operation are determined empirically depending upon the desired final or maximum aspect ratio of the intermediate shell product for any particular glass composition. 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 2.3 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 (1 5OWC), were in the size range of 90 to 180 microns. The intermediate product shells in this example had a size range of 75 to 250 microns and an aspect ratio of 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 tailoring operation. Specifically, the shells 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 desired size, in this case 106 to 200 microns, and immersed in a second solution having a density 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 invention to reduce the density thereof to 1.04 g/cc. More specifi- 3 GB 2 15161 OA 3 cally, 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 discarded. The floatersin carbon tetrachloride are then immersed in a solution of 15% sulfuric acid (1.10 g/cc) and 4% - hydrogen fluoride, the latter being an etching agent. As the shells become buoyant, indicat ing removal of surface glass and density de cline to 1. 10 g/cc, they are removed and immersed in a solution of 5% sulfuric acid (1.04 g/cc) and 2% hydrogen fluoride.

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 fibrosar coma and Walker carcinosarcoma,in RPM[ media with 10% fetal calf serum. The micro carrier shells are substantially buoyant- in the culture medium and may be readily main tained in suspended state by mild agitation, such as by-mild aeration using carbon dioxide bubbles which are otherwise useful to control 95 medium pH. The glass shell microcarriers may be treated with amine salts for forming sur face charge moieties, although this is pre sently believed to be unnecessary. The shells may also be readily coated with a desired material using conventional techniques. Of course, the thickness and density of any coat ing must be taken into consideration during the density tailoring operation..

In high-volume production of glass shell microcarriers in accordance with the inven- tion, it is anticipated and contemplated that the various process steps hereinabove de scribed be fully or at least partial ly'auto mated.

For example, skimming apparatus may be associated with each culling or etching stage for automatically removing floaters. Depend ing upon accuracy of control during the vari ous operations and tolerance of desired final density range, the two-step etching operation herein described by way of example may be replaced by one stage, or for that matter.

increased to three or more stages. Strength of etchant in solution, and therefore required etchant time, was selected in the example for 120 best batch control, and may vary depending upon circumstances. 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 125 exemplary -1.0 g/cc to 1.04 g/cc range de scribed herein. For example, if it were desired to produce shells having densities closely clus tered about 1.04 g/cc, the initial culling step in water could be skipped, and the intermei- date shell product could be immersed in the 5% aqueous sulphuric acid solution. Floaters, having a density below 1.04 g/cc would be discarded 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 of narrowing 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 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 culturation. See, for example, Wehrenberg et a[, -Shedding Pounds in Plas- tics:. Microspheres are Moving,- Mechanical Engineering, October 1978, pages 58-63. In this respect,4 the density of the final shell may vary widely-from the exemplary range of 1.0 to, 1.04-g/cc, and also from the range of 1.03 to 1.09 g/cc for typical cell culture media. 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 parameters of the gel out-gassing operation (which decreases shell aspect ratio) are determined empirically based upon desired aspect ratio fol]owing the'biowing operation, which in turn is determined mathematically based upon den- sity of the glass composition employed and desired final.shell density and size.

The invention claimed is:

Claims (9)

CLAIMS --,----; 1

1. A hollow midrosphere consisting of a closed shell of integral and essentially homogeneous silicate,glass composition having a density in excess of one gram per cubic centimeter.

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2. A'microcarrier adapted for use as growth sites for anchoragedependent cells in a cell culture medium of predetermined density comprising a hollow spherical shell having a homogeneously integral and continuous shell wall of silicate glass composition and a density substantially equal to said predetermined density.,.

3.. The microcarrier set forth in claim 1 or 2 wherein. said density of said shell is in the range-of 1.03 to 1.09 g/cc.

4. - The microcarrier set forth in any of claims 1 to 3 wherein said shell has an aspect ratio of outside diameter to wall thickness no greater than 12. ---

5.- A method of growing anchorage-dependent cells in a cell cultijre medium of predetermined' density by employing a multiplicity of microcarriers i n the culture medium as cell anchorage sites- in which the microcarriers are compfised of Cilow spherical shells of essen- 4 c GB 2151 61 OA 4 tially homogeneous silicate glass composition and having an average shell density substan tially equal to said predetermined density.

6. A method as claimed in claim 5 in which the predetermined density is in excess 70 of one gram per cubic centimeter and in which said microcarriers comprise a multipli city of hollow spherical shells of essentially isotropic silicate glass composition having an average density, determined by shell compo sition, wall thickness and diameter, substan tially equal to said predetermined density.

7. A process for manufacture of hollow silicate shells from a metal organic gel com prising the steps of: (a) preparing a metal organic gel to include glass-forming metallic components and a blowing agent, (b) crushing said gel to form gel particles, and (c) subject ing gel particles to elevated temperature above the softening temperature of said glassforming components to promote volatilization of said blowing agent to form said particles into hollow spherical shells, in which prior to said step (c) said gel particles from said step (b) are subjected to an out-gassing operation by melting said particles to drive off a portion of said blowing agent and form an intermedi ate foam-like aggregrate, said foam-like aggre gate is crushed to reform individual particles, and the reformed particles when subjected to 95 said step (c) whereby the aspect ratio of diameter to wall thickness is reduced and the density of said shells thereby increased.

8. A method of adjusting the density of preformed hollow microspheres having a pre formed density greater than a desired final density comprising the steps of subjecting said preformed microspheres to a surface etching operation in a solution having a den sity equal to said desired final density and removing said microspheres from said solution when said shells become buoyant.

9. A glass microsphere comprising a homogeneously integral and continuous spherical shell manufactured in accordance with the method claimed in any of claims 1 to 8 having a density in excess of one gram per cubic centimeter.

Printed in the United Kingdom for Her Majesty's Stationery Office, Dd 8818935, 1985, 4235. Published at The Patent Office, 25 Southampton Buildings, London. WC2A 'I AY, from which copies may be obtained.

1 1

9. The method set forth in claim 8 wherein said solution comprises an aqueous solution having a said density, equal to said 110 desired final density, in excess of one gram per cubic centimeter.

10. The method set forth in claim 8 or 9 wherein said preformed microspheres are of silicate glass composition.

11. A method as claimed in any of claims 8 to 10 in which the preformed hollow microspheres have been produced by a method comprising the steps of: (a) forming a metal organic gel which includes glass-forming components and a blowing agent, (b) drying and crushing said gel to form a multiplicity of gel particles, (c) expanding said shells in a blowing operation to form hollow spherical shells, at least a portion of which have a density in excess of said desired final density.

12. The method set forth in claim 11 comprising the additional step prior to said etching step of: subjecting said hollow spheri- cal shells to a culling operation so as to 130 separate said portion of said shells, whereby only that portion of said shells having a density in excess of said desired final density are subjected to said etching step.

13. The method set forth in any of claims 6 to 12 wherein said desired final density and said density of said solution is in the range of 1.03 to 1.09 g/cc.

CLAIMS Amendments to the claims have been filed, and have the following effect:- Claims 1 - 13 above have been deleted or textually amended.

New or textually amended claims have been filed as follows:- 1. A method is provided of adjusting the density of preformed microspheres comprising homogeneously integral and continuous spherical shells having a preformed density greater than a desired final density comprising the steps of subjecting said preformed microspheres to a surface etching operation in a solution having a density equal to said desired final density and removing said microspheres from said solution when said shells become buoyant.

2. A method as claimed in claim 1, wherein said solution comprises an aqueous solution having a said density, equal to said desired final density, in excess of one gram per cubic centimeter.

3. A method as claimed in claim 1 or 2 wherein said preformed microspheres are of silicate glass composition.

4. A method as claimed in any of claims 1 to 3 comprising the steps of: (a) forming a metal organic gel which includes glass-forming components and a blowing agent, (b) drying and crushing said gel to form a multiplicity of gel particles, (c) expanding said shells in a blowing operation to form hollow spherical shells, at least a portion of which have a density in excess of said predetermined desired density, (d) subjecting said portion to an etching operation by immersing said portion in an etchant solution having a density equal to said predetermined desired density, and (e) removing said shells from said solution when said shells become buoyant in said solution.

5. A method as claimed in claim 4 comprising the additional step prior to said step (d) of: (f) subjecting said hollow spherical shells to a culling operation so as to separate said portion of said shells, only that portion of said shells having a density in excess of said predetermined desired density being subjected to said steps (d) and (e).

6. A method as claimed in claim 4 or 5 wherein said solution is an aqueous solution having a density in excess of one gram per cubic centimeter.

7. A method as claimed in claim 4, 5 or 6 which, in order to reduce the aspect ratio of GB 2 15161 OA 5 diameter to wall thickness and thereby increase the density of said shells, comprises the additional steps prior to said step (c) of: (x) subjecting said gel particles from said step (b) to an out-gassing operation by melting said particles to drive off a portion of said blowing agent and form an intermediate foamlike aggregate, (y) crushing said foam-like aggregate to reform individual particles, and then (z) subjecting said reformed particles to said step (c).

8. A method as claimed in any of claims 1 to 7 wherein said predetermined desired density and said density of said solution is in the range of 1.03 to 1.09 g/cc.