GB2338243A - Macro carrier for cell culture - Google Patents

Abstract

A transparent multilayer carrier is fabricated from a plurality of regularly and closely spaced plastic foils. The spacing may be of the order of 20-100 microns to allow cells and growth medium to move unimpeded laterally between the layers. The foils may be microperforated to allow transverse permeation of cells and medium. The multilayer supports may be manufactures by piercing spaced layers of thermoplastic film with a hot vibrating needle which generates fibrous struts between the layers, or the layers may be separated by conical rims which project from perforations. The macro carrier may be less dense than water so that in use it floats.

Description

1 2338243 - 1 TRANSPARENT MACRO CARRIER FOR CELL CULTURE

TECHNICAL FIELD

The invention relates to cell and tissue culture; specifically, to a carrier surface for bulk culture, microscopic inspection, harvesting and cloning of anchorage dependent animal cells.

BACKGROUND

In vitro culture is carried out by seeding cells into a vessel together with a liquid medium for provision of chemical components. But socalled "anchorage dependent" cells biologically obligated to attach and grow on a surface require an additional mechanical component: the support surface. Other types of cell may also be immobilized on a carrier support for convenience. In such cases the number of cells cultured is directly related to surface area provided.

We11known methods of surface treatment can promote adhesion of cells to transparent plastics. We shall disclose methods for fabrication of novel transparent multilayer Macro carriers from readily available films of surface treatable plastic films.

Two we11known culture vessels, T-flask and roller bottle, though conveniently transparent, have so little wall surface that the number of cells grown and hence the volume of medium processed, is typically only 10% of their full volume capacity. An accordian pleated wall to increase the surface of a roller vessel is described in US Pat 5 010 013; and pleated wall T-flasks have near double capacity. In contrast, we shall disclose a device that at least quadruples cell production capacity in an ordinary T-flask without pleats.

Cells can be anchored to an outer surface on nonporous carriers - for instance, microcarrier beads in stirred vessel, or twisted ribbons in packed column (US Pat. 5 168 058). But the disadvantage of solid carriers is, that cells may be damaged when carriers are stirred or packed together. In contrast, present invention is a novel version of a type, which we may call a macroporous Macro carrier ie, a carrier which is both larger than typical microcarriers, and also has pores large enough for cells to shelter inside.

Macroporous Macro carriers have been made from various materials. Looby and Griffiths, in Ann. NY Acad Sci. 1992, Oct.13, vol. 665, p.146-151, describe sintered glass beads, diam 2mm-5mm. EP 546 144A1 930 616 describes porous plastic spheres weighted with ceramic or metal powder to a specific gravity of 1.15. Israel patent 75554 describes discs of 1 1 1 1 2 - nonwoven polyester fibers with a pore size of 10 to 100 microns, disc thickness from 50 to 500 micron, diam 6mm. Nilsson et al, in Cytotechnology 1990 3: 271-277, describe microcarriers of macropous gelatin; and US Pat 5 100 783 describes small spheres of weighted collagen gel. Said macroporous granular or fibrous or gelatinous matrices have internal channels with a random distribution of size, shape and orientation: consequently, cells remain trapped in their tortuous interstices, hence cannot be harvested with acceptable efficiency by the normal method of detachment with trypsin enzyme. Cells can be harvested by chemical dissolution of a gel matrix; but since said matrix is thereby destroyed, this method of harvest is a once only process. In contrast, the present invention is a regular structured matrix whose pores are planar channels, whose pore dimensions are reasonably uniform and larger than the cell diameter, and whose walls are uniformly perforated to allow additional egress for cells in the transverse direction, so that cells can be efficiently harvested with trypsin, and the carrier can be reused to yield multiple harvests.

Moreover, the same improved features of our invention - wide, planar channels of uniform height, whose open mouths debouch directly into the surrounding medium, plus transverse permeability - are also features which increase the flow of medium through the multiplanar carrier of present invention (or which allow the same flow but with less stirring or a lower pressure drop) as compared with aforesaid random granular, fibrous or macroporous gel matrices with their narrower channels and more tortuous flowpaths.

Lastly, random-pore carriers tend to opacity through internal light scattering, which impedes the inspection of cells by conventional light microscopy. In contrast, the regular pore structure with parallel planes of present invention has much improved transparency. The single plane carrier cited above (US Pat 5 168 050) was equally transparent but multiplane of present invention offers improved protection of cells. Also mentioned above, the transparent T-flask is convenient both for microscopic inspection of the culture in situ, and for optical screening of individual cells or colonies in a selection process known as cloning; but inadequate wall surface limits the initial population of cells and hence the number of candidates for selection. The use of transparent multilayer carriers, in a T-flask, not only retains most of that flask's convenience of optical inspection in situ, but provides the additional convenience of withdrawing selected colonies, on our Macro carriers, for subculture or histochemical inspection.

The use of multiplanar transparent plastic film or sheet in cell culture is not new: for instance, the spiral film described by Maroudas in Methods in Cell Biology, vol. 8, 1974; or the rectangular multideck vessels marketed as "Multitray by Nunc and "Cellcubey, by Costar. But these multilayer devices have taken the form of a large module - usually an integral structural member - fixed in a made-to-measure housing; their interlayer spacings are correspondingly large; and these multilayers are not uniformly microperforated. In contrast, present invention allows manufacture of much smaller multilayer elements, which are freely movable and randomly packable, hence can be used in a variety of freely chosen flasks, columns or stirred vessels, without the aforesaid constraint of housing a unique large carrier module. The Macro carriers of present invention are at least ten times smaller than above cited modules (largest dimension 8-12 mm, versus 150-300 mm). Their interlayer distance is at least 10 times closer and hence more space saving (0.02-0.1 mm, compared with 1-10 mm).

The novelty of present invention lies also in the use of uniformly microperforated film, which supplies an alternative cross-route for cells, fluid and diffusible metabolites to permeate the parallel channels to compensate for closer spacing in present invention.

- 4 ESSENTIAL TECHNICAL FEATURES According to the present invention there is provided a stack of transparent film, in a regularly spaced multilayer structure of parallel foils that acts as a support surface for attached cells. The minimum spacing between layers must be large enough to allow both the cells and their fluid medium to move reasonably unimpeded laterally ie, in the flat channels parallel to the layers, 1. An optimal spacing for animal cells is 20-100 micron: smaller impedes lateral movement, larger wastes space.

In a preferred embodiment of present invention, said multilayer foils are also microperforated, with holes, 2, uniformly distributed to allow both the cells and their fluid medium to permeate transversely ie, to move in a reasonably uniform manner perpendicular to, and through, the foil layers 1, not restricted to lateral movement as above. A practical hole size and density, from readily available flame perforated film, is 500 micron diam and 50 holes per square cm, but perforation by some other process to produce smaller holes (e.g 20 micron for animal cells, or 1 micron for bacterial cells) or more holes per unit area, would still exemplify the present invention of putting microperforations into transparent multifoil supports that allow transverse permeation by cells as well as fluid. If assembled into narrow (0.5-2mm) rectangular Macro carriers, which have a short path for diffusion of oxygen edgewise, such multilayers need not have transverse holes. Conversely, said microperforations are an important aid for efficient diffusion of metabolites and movement of cells in closely spaced multilayers (100 micron spacing or less) if said layers are required to be broad (above 5 mm width) as well as long.

1 The use of polyolefine films, which are less dense than water, allows the carriers to float at the air / water interface of the nutrient medium, and hence allows the cells to lie within the oxygen-rich liquid diffusion boundary layer, which is about 2 mm thick. If the floating multilayer carrier has been correctly designed, as above, with good permeability edgewise and/or transversely, then cells can metabolize by diffusion alone, with little or no stirring of the medium. However, other films, with a range of specific gravities can be also be used (e.g., polystyrene, polyester and their composites with polyolefine) to make carriers of different specific gravity: for instance, denser than water to settle down when packing a column; or with neutral buouyancy (equal to that of medium)) to remain in suspension with little or no stirring.

FIGURE 1 is a three dimensional perspective of a preferred rectangular embodiment of the cell culture support in which the layers L are joined by strut fibres 3, transverse to the plane of the layers and situated at the periphery of some of the holes 2. Such fibrous struts are made as follows, by a process similar to sewing or needle felting. Horizontal multilayers of thermoplastic film are held at the predetermined spacing while a hot needle, vibrating in the vertical direction, simultaneously pierces the thermoplastic foils and draws out molten fibres which solidify to form struts. The assembly is then cut into individual carrier particles. With spacings of 20-100 micron between layers and 2-5 mm between struts, Macro carrier assemblies can be made which are reasonably rigid for use in stirred vessels or packed columns, in a size preferably 5 mm long by 0.5-2 mm wide and with a thickness of 5-20 layers.

FIGURE 2 is a crossection of the embodiment of FIG. 1 showing how the fibres, 3, pierce through the layers, 1, and join them together, through holes which are aligned by the passage of said hot needle.

In both figures 1 and 2, it is understood that the holes, 2, in said foils, 1, can be of two sorts: holes made by said hot needle process, aligned and joined by said fibrous struts, 3; and optional additional holes without struts, because said needling can optionally be performed on ready perforated film. In the latter case, the additional holes need not pierce through every layer inline as in fig 2, but can be staggered in different layers. The use of ready perforated film gives us the option to space and join the layers by other means than by said perforating struts.

FIGURE 3 is a three dimensional perspective of an embodiment which uses film with readymade potruding conical perforations. Each perforation consists of a hole, 2, at the summit of a conical rim, 4; such shapes (similar to a volcano) are manufactured by the we11known "flame perforation" of film. We can exploit the readymade conical rims, 4, and produce self spacing layers, as follows.

FIG. 4 is a crossection of a rectangular, raft shaped carrier made from conically perforated foils as in FIG. 3, held together by a single join, 5, at the center (e.g. spot weld).

FIG. 5 is a crossectional of sheets similar to FIG. 4 but with two joins, 5, one at each end of the carrier's length.

In both figures 4 and 5, the holes are understood to be of conical shape as in FIG. 3, in order to make a self spacing multilayer. Holes are staggered in each alternate layer, so 6 - that the conical projections, 4, do not nest inside one another but act as separators. In each layer the hole mouths, 2, at the rim of cones, 4, are wholly or partially blocked by a staggered counterlayer, hence movement of fluid and cells will be mainly edgewise, through the open peripheral mouths of the parallel multilayer channels. Consequently, the preferred embodiment of figures 4 & 5 is a narrow (0.5-2 mm) rectangular carrier so that each foil exposes two long edges and has a short flowpath across its width.

Although the preferred embodiment of FIGS. 3, 4 & 5 show foils made from readily available film with conical holes of fixed size and spacing, special patterns can be made: for instance, conical dimples (without holes) to improve spacing and hence edgewise permeability, plus planar holes (without projecting rims) to improve transverse permeability.

REASONS FOR SOME PREFERRED EMBODIMENTS The embodiments of Fig.1 and Fig.5 are preferred for use in packed columns, since these more rigid structures resist mechanical compaction under pressure of pumping liquid. The more open, butterfly structure of FIG. 4 may be more suitable for use in static culture flasks or gently stirred vessels.

Reasons for preferred dimensions as a long flat rectangular carrier with 5-20 decks, 0.5-2 mm in width and/or height, and length 5-15 mm, are as follows.

1. The number of layers of film is a compromise between the need to minimize the proportion of the two outer surfaces (where cells can be damaged as described above) versus the need to preserve transparency. optimally, 5-20 layers would protect 80-95% of the cells while maintaining good transparency. Preferred thickness of a multilayer assembly with average spacing 100 micron is thus 0.5-2 mm.

2. Preferred width in the plane of the channels depends on the intensity of convective flow (pumping or stirring). In the worst case, of static culture and edgewise diffusion only, width should be less than 2 mm, to allow viable penetration of oxygen (for 2 mm limit see e.g., Folkman & Greenspan, 1975).

3. The minimum spacing between layers depends on the size and stickines of given cells, and whether there is need to harvest; most animal cells have diameter 5-20 micron, but other cells and / or smaller spacing would still exemplify the multilayers of present invention so long as they allowed reasonably free ingress and egress of cells and fluid for growth and harvest.

7 - Because both sides (top and bottom) of each foil are available for cell adhesion and growth, the maximal available culture surface is twice the number of stacked foils. For example, with 100 um spacing hence 100 stacked planes per cm of height, maximal surface would be 200 sq cm per cm cube, or 1,000 cm2 per gram of polypropylene film 20 micron thickness. At 2 E5 cells per square cm, maximal cell density would be 4 E7 cells per cm cube carrier volume or 2 E8 cells per gram. Actual cell density may be less than the maximal, due to uneven coverage and growth. Loose packed, 1 gram of said multiplane Macro carriers would occupy about 8-15 ml of volume and present the surface equivalent of a 2 litre roller bottle (800 cm2).

4. Length of the carriers should be preferably less than one tenth the cross section of the vessel, to allow good packing or stirring: for instance, 5-15 mm length in 5-15 cm vessels.

5. The long, narrow carrier shape of present invention also has a hydrodynamic advantage over spherical carriers: the shape provides an increased lift force, hence said Macro carriers can be used at low stirring speed (30-300 rpm) in spite of their comparatively large size (515 mm long, compared to 0.13-0.25 mm diam for spherical microcarriers). Larger size provides the advantage of rapid and easy separation of Macro carriers from liquid by means of a coarse sieve, 15 mm mesh.

EXPERIMENTAL EXAMPLES OF USE THE EXPERIMENTS were conducted on 2-4 gram samples of carriers with 6-20 layers, 6 to 12 mm in length and 0.5-3 mm width, made from autoclavable polyolefine film. The carriers were used inside three kinds of culture vessel. The first was an old fashioned glass Roux bottle in static culture. The second kind was a set of modern disposable plastic T-flasks, volume 600 ml and area 150 cm2. This size flask in normal use typically yielded us about 15 million cells, which process only 30 ml of culture medium ie, 95% of the flask volume is unused. Addition of floating multiplanes increased the cell yield fourfold, and allowed 200 ml of medium to be processed in static culture. The last kind was a stirred vessel.

METHOD - CELLS AND CONTROL TRAY CULTURE CHO strains, transfected with IL6 receptor, were maintained in DMEM/F12 medium with 10% Fetal Calf Serum. Confluent cultures reached 5 million cells per 9 cm dish (0.8 E5 cells/cm2). Trypsinised cells were counted by hemocytometer with Trypan blue (95% viable cells).

8 Control production was on 600 cm2 trays which occupied about 1 litre in volume, 90% of which was empty space. Cells were seeded at 5 million per tray in 50 ml of medium with 10% FCS. At confluence (40-50 E6 cells/tray) the medium was changed to 2% FCS, harvested and replaced with a fresh batch of 2% medium every 4 days until the cell culture gave out (about 4 batches in tray culture).

METHOD - CELL SEEDING ON CARRIERS IN FLASK Seed cells are applied in the minimum of excess medium. The interior liquid of the Macro carrier is about 1/4 of the bulk volume. To 20 ml of carrier in a T150 flask were added 5-10 ml of inoculum (10-25 E6 cells) and gently mixed with carriers by tilting the flask. Dry Macro carrier soaked up this volume of medium completely. If the Macro carrier had been washed (ie, was already holding 5 ml of cellfree medium) then the flask would be gently tilted to mix in the excess medium. The flask was laid flat in incubator; every 1/2 hour it was examined under microscope for cell adhesion and speading, and gently rocked to redistribute nonadherent cells.

METHOD - CELL GROWTH IN FLASK Incubate in C02 atmosphere to start. Examine under microscope for growth. At confluence, the cells may generate enough C02 to warrant incubation in air. Use a vented cap of 1-2 mm aperture. Increase the amount of medium to 100-300 and the aperture of the vented cap, according to cell growth and metabolism. If rocked, once per minute is sufficient.

METHOD - HARVESTING CELLS FROM MULTILAYER CARRIERS Trypsinize as for normal dish culture, with amount of trypsin increased pro rata with number of cells. Wash cells out of carrier by gently shaking the flask. Separate cells from carriers through 1-2mm pipette nozzle or coarse sieve.

METHOD HARVESTING OF SOLUBLE PRODUCTS A 2 mm mesh strainer, in the neck of the T-flask, retained the Macro carriers while the medium was being poured in or out of the flask as normal.

METHOD MICROSCOPY For unstained bulk cells in situ, the culture flask was placed on a conventional inverted microscope; X2.5 to X20 objectives could focus on carriers floating on a 6-10 mm layer of medium. Sample Macro carriers were withdrawn with forceps or spatula, and stained in a well, washing with a bulb pipette. Confluent multifoils became opaque with absorption stains like Giemsa, but could be cut with scissors to observe cell sheets on individual foils. Selective fluorescescent stains like Acridine Orange pick out cells through successive multilayers.

RESULT - CELL PRODUCTION IN T-FLASK A T150 cm2 flask with vented cap, 100 ml of medium, 5% FCS and 20 ml Macro carrier was seeded with a single inoculum of 10 million CHO cells. Near confluence the flask was placed on a slow rocker and trypsinised weekly for a month, to yield four successive harvests of CHO cells. (Each New harvests was generated from residual cells of the previous harvest).

Cell yield from flask surface alone:

million Cell yield from flask plus Macro carrier: 60 million Total cell yield after four harvests: 240 million RESULT - IL6 RECEPTOR YIELD IN ROUX BOTTLE A 1 litre glass Roux bottle was sterilized by autoclave after inserting 4 gram of carriers with fibre struts as in FIG. 1. The flask was seeded with 15 million cells and laid flat in a C02 incubator. At confluence the serum was lowered to 2%. The floating carriers were left in static culture for 7 weeks, and batch fed twice weekly by gentle decantation of the conditioned medium and replacement with fresh of 2% medium. Control cultures in 600 cm2 trays did not last more than 2 weeks.

ML/BATCH TITRE TOTAL TOTAL YIELD BATCHES PER VESSEL ug/M1 mg CONTROL TRAY 75 3.1 4 0.93 ROUX BOTTLE 20-250 1.4 15 4.80 Although product titre was lower in this case, total yield of product was greater, because the Macro carrier culture lasted longer and processed more medium, in a vessel with the same 1 litre volume as the control tray.

RESULT - IL6 RECEPTOR YIELD IN T-FLASKS A set of 20 Trays was compared with a set of six T150 flasks with 1.8 gram (15 ml) of carriers, assembled from film with conical holes as in FIG 4. Trays and flasks alike were static incubator culture. The tray cultures lasted long enough for us to harvest 3 batches of 75 ml per tray, and the T-flasks yielded 9 batches of 200 ml per flask.

MI, PER TITRE TOTAL BATCH BATCHES CONTROL TRAY 75 ug/M1 5.0 3 TOTAL YIELD mg/vessel 1.125 T150 FLASK 200 7.0 9 12.6 - In this case, the titre was also greater which, in combination with the above mentioned increase in culture volume and life, resulted in a tenfold increase in total yield.

RESULT - IL6 RECEPTOR PRODUCTION IN SPINNER VESSEL Four gram of sterile multilayer carriers, in 50 ml of 5% serum medium, were poured into a 1 litre spinner vessel through one of its two 1" sideports. A 1" by 2 mm mesh strainer was push fit in the neck of the other port. Changes of medium were effected by pouring through this strainer port, while samples of Cellraft were removed with a long spatula through the free port.

After seeding with 20 million cells, 300 ml of medium was added - enough to lift the floating carriers well clear of the stirrer, which was set 3060 rpm, just enough to mobilize the carriers but not enough to drag them into the vortex around the stirrer blades.

At confluence, medium was changed to 2% serum and titre assayed every 4 days. Three batches of medium were harvested. No problems were encountered with cells stripping off the carriers. The culture also recovered from an initial stoppage of the stirrer motor overnight, probably because the cells in our floating carriers could obtain oxygen by diffusion from the liquid boundary layer, as explained above.

ML/VESSEL TITRE TOTAL TOTAL ug/M1 BATCHES YIELD mg/vessel SPINNER VESSEL 300 3.3 3 2.97 4 1 g 1 RESULT - MICROSCOPY Unstained individual cells in bulk culture were clearly visible through 6-10 successive layers. On fluorescent stained sample carriers it was easy to focus through the multilayers onto individual cells. Acridine Orange fluorescence showed many cells with active nuclei, some with chromosomes and mitotic bodies clearly visible. With absorption stains, examination of individual foils clearly revealed nuclear and cytoplasmic granules; and also confirmed that cells were growing on both the upper and the lower side of the film layer.

RESULT - COLONY SELECTION The transparency of multilayer carriers in a Tflask was good enough for us to observe a small number of remaining single cells or colonies that survived - attached within the raft multilayers after a serumfree selection that destroyed and detached the vast majority of cells out of an initial population of 60 million in 10% serum.

11

Claims (9)

1. A stack of transparent film, in a regularly spaced multilayer structure of parallel foils that acts as a support surface for attached cells. The minimum spacing between layers must be large enough to allow both the cells and their fluid medium to move reasonably unimpeded laterally ie, in the flat channels parallel to the layers, 1. An optimal spacing for animal cells is 20-100 micron: smaller impedes lateral movement, larger wastes space.

2. multilayer foils according to Claim 1, that are also microperforated, with holes, 2, that allow both the cells and their fluid medium to permeate transversely ie, to move in a reasonably uniform manner perpendicular to, and through, the foil layers 1, not restricted to lateral movement as above. A practical hole size and density, from readily available flame perforated film, is 500 micron diam and 50 holes per square cm, but smaller holes (e.g 20 micron for animal cells, or 1 micron for bacterial cells) and more holes per unit area, would still exemplify the present invention of putting microperforations into a multilayer support surface, so as to allow transverse permeation by cells and fluid.

3. An assembly of transparent multilayers of film according to Claims 1 & 2, in the preferred form of flat, narrow rectangular, freely movable Macro carriers, of dimension typically 0.5-2 mm wide and 515 mm long, with 5-20 layers spaced typically 20-100 micrometre apart.

4. Multilayer supports of Claims 1-3 which are manufactured by piercing spaced layers of thermoplastic film with a hot vibrating needle that generates fibrous struts, 3.

5. Multilayer Macro carriers of Claim 3 which are manufactured from foils with a staggered self spacing pattern of dimpled holes with conical projecting rims, 4, fixed together by one or two localized joins, 5.

6. Multilayer supports according to Claims 1-5, which are self spacing by a staggered pattern of conical dimples, 4, but the dimples are blind i.e. without holes, 2.

7. Floating carriers according to Claims 16, made from transparent film less dense than water, which float within the oxygen rich, liquid diffusion boundary layer at the air/water interface of the culture medium.

8. Multilayer carriers of Claims 1-6, made from film materials or composites with a designated specific gravity equal to, or greater than, that of the liquid medium.

9. Use of a transparent multilayer cell carrier for optical screening operations on a large population of cells.