EP0498858A1

EP0498858A1 - Nested-cone separator

Info

Publication number: EP0498858A1
Application number: EP91900644A
Authority: EP
Inventors: William G. Thilly; Michael A. Tyo
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 1989-11-03
Filing date: 1990-11-02
Publication date: 1992-08-19
Also published as: CA2072141A1; WO1991006627A1; JPH05504054A

Abstract

Un système de culture de cellules permet de séparer des cellules d'une substance de perfusion dans une culture de cellules. La substance de perfusion et les cellules en suspension dans la substance de perfusion passent à travers un séparateur à cônes emboîtés ayant un cône externe et un cône interne qui définissent un espace annulaire à section transversale d'écoulement en expansion, de sorte que la vitesse de la substance de perfusion dans l'espace annulaire diminue à mesure qu'elle s'éloigne de la culture de cellules en suspension, ce qui permet de réduire la vitesse superficielle de la substance en perfusion. Un effet de Boycott et une vitesse superficielle réduite dans l'espace annulaire permettent aux cellules qui doivent revenir à la culture de cellules en suspension de se déposer le long du trajet d'écoulement de la substance de perfusion dans l'espace annulaire et de revenir à la culture de cellules. La substance de perfusion se sépare ainsi des cellules et est éliminée de la culture de cellules. En même temps, une commande très fiable et efficace du point de vue économique des cultures de cellules est assurée pendant de longues périodes.A cell culture system separates cells from a perfusion substance in a cell culture. The perfusion substance and the cells suspended in the perfusion substance pass through a nested cone separator having an outer cone and an inner cone which define an annular space of expanding flow cross-section, so that the speed of the perfusing substance in the annular space decreases as it moves away from the cell suspension culture, allowing the superficial velocity of the perfusing substance to be reduced. A Boycott effect and reduced superficial velocity in the annular space allow cells that need to return to the suspension cell culture to settle along the flow path of the perfusion substance in the annular space and back. to cell culture. The perfusion substance thus separates from the cells and is removed from the cell culture. At the same time, very reliable and economically efficient control of cell cultures is ensured for long periods of time.

Description

NESTED-CONE SEPARATOR Background of the lnvention

Cell culture systems wherein cells are suspended within media are a cost-effective alternative toin vivo manufacture of cell products. The behavior of suspended cell cultures is often dependent upon cell concentration. Limitations to high cell concentration include the ability to remove waste, toxins, dead cells and cell products from the cell culture system. Accumulation of toxins produced in the cell culture, for example, can terminate cell production. In addition, where such systems are to be maintained for long periods of time, cell

products must be collected by methods which do not disturb conditions within the cell culture.

Common to problems with conventional methods for removing fluids from suspended cell cultures is that suspended cells are often only slightly more dense than the media by which they are supported. Suspension of such cells is maintained by stirring the media and cells. Separation techniques which remove or isolate large fractions of cells in a culture from the oxygen or nutrient source wi thin the system often diminish productivity, particularly of perfusion and circumfusion cell culture systems. Further, cell viability, which can be defined as the fraction of cells in the culture having the ability to reproduce by cell division, can be contingent upon rapid removal of cells, cell products and waste-containing perfusate.

One method of separation has been isolation of volumes appurtenant to the suspension vessel for settling of cells from perfusate. Settling devices have included columns and funnels. See e.g., H. Murakami et al., Proc. of the International Sym.,

Growth and D ifferentiation of Cells in Defined

Environment, Springer-Verlag (1985); K. Kitano et al., "Production of Hunan Monoclonal Antibodies by Heterohybridomas," Applied Micrgbiρlogy and Biotechnology, 24 282-286 (1985); and J. Tabera et al., "Design of Lamella Settler for fiiomass Recycling in Continious Ethanol Fermentation Process,"

Biotechnology and Bioengineering, 33: 1296-1305 (1989).

Murakami et al. teach a sedimentation column which is partially submerged within a cell culture system. An impeller is fixed to the submerged end where perfusate and suspended cells enter the column and settle along the interior of the column.

Perfusate is removed from the column at a point elevated above the cell culture. Kitano et al.

teach a cell precipitator suspended above a jar fermenter. The precipitator has a lower funnel portion and an upper column portion. Cells settle within the precipitator and travel back to the fermenter, while perfusate is drawn by a pump from the top of the precipitator. Another separator, described by Tabera et al., is a chamber having parallel plates disposed therein at an acute angle to the horizontal. Cell culture media from a fermenter enters at the plates. Laminar flow between the plates causes cells to collect along the plate surfaces and to continuously deposit at the bottom of the chamber for pumping back to the fermenter. Despite these prior techniques, the effect of high rates of removal from cell cultures and the consequent effects on cell viability, have limited utility of perfusion and circumfusion cell culture systems. In addition, continuous settling of cells generally has required occupation of a large

fraction of the cell culture system, thereby

isolating a large portion of cells from the cell culture system, and often limiting productivity.

Thus, there is a demand for a system for separation of perfusate from cells which can rapidly remove harmful by-products from cell culture systems while maintaining high levels of cell viability.

Summary of the Invention

This invention relates to a cell culture system, a nested-cone separator and a method for separating perfusate from a cell culture. A

nested-cone separator has an outer cone and an inner cone nested within the outer cone. The narrowest portion of the outer cone defines an inlet port which is submerged in the cell culture. The outer and inner cones define an annulus forming a cross- sectional area for flow which expands with increased distance from the inlet. An outlet port is elevated from the inlet port at a head space above the inlet port. The annulus provides fluid communication between the inlet port and the outlet port.

A method for separating perfusate from a cell culture includes flowing perfusate and cells from a cell culture through a nested-cone separator. The perfusate is conducted through an expanding annulus of flow, which is defined by an outer cone and an inner cone. The "superficial," or vertical, velocity of the perfusate is diminished in the annulus and cells within the perfusate consequently settle across the path of perfusate flow and collect at an inner wall of the outer cone.

The appropriate width between the walls of the annulus and the distance between the inlet port and the outlet port can be determined by the "Stoke's Law terminal velocity" of the cells, which is defined as the velocity of vertical perfusate flow, below which cells will settle from the perfusate, and by the "perfusion rate," which is defined as rate at which perfusate in the suspended cell culture is replaced with fresh media. The path of perfusate flow from the inlet port through the expanding annulus is divergent, thereby reducing the superficial velocity of flow with increasing

distance of perfusate from the inlet. The resultant pattern of flow of perfusate in the annulus is such as to allow cells to settle across the path of perfusate flow. The cells in the perfusate thus collect along an inner wall of the outer cone.

Perfusate, comprising desired product, cells, undesired waste, and toxins, is thereby separated from cells to be returned to the cell culture and is removed from the annulus at the outlet port. Cells accumulated at the inner wall of the outer cone are directed by gravitational force along the inner wall, opposite the direction of flow of the

perfusate, and re-enter the cell culture at the inlet port.

The nested-cone separator of the present invention is particularly advantageous for separation of perfusate from cultures of anchorage- dependent cells grown on microcarriers. The Stoke's Law terminal velocity of anchorage-dependent cells can be raised by adherence of the cells to

microcarriers. The same effect occurs where

anchorage-independent cells adhere to each other, as in flocculence. In the case of either

anchorage-dependent cells or in flocculence, the efficiency of separation by the present invention of perfusate from cells is thereby increased.

The suspended cell culture system of the present invention allows effective and controlled separation of perfusate from a wide variety of cell cultures. The present invention can be used, for example, for manufacture of monoclonal antibodies, viruses, Sp2/0-derived transfectoma cells, hormones and other cell-derived macromolecules, and cells for use in humans and other organisms. The present invention can also be used to maintain cell cultures for repetitive sampling of cells, antibiotic

fermentation, and as an alternative to sedimentation in sewage treatment.

The nested-cone separator enhances a Boycott effect, wherein cells settle within an inclined conduit faster than they do in a vertical conduit. Diminished superficial velocity, caused by the expanding annulus, and the Boycott effect allow cells, which are to be retained within the cell culture, to settle across the path of perfusate flow and to collect at an inner wall of the separator for return to the cell culture. Further, rapid

separation of cells to be returned to the cell culture from perfusate in the separator

substantially reduces the period of isolation of cells from the culture and nutrient and oxygenation sources of the cell culture system. Thus, larger cell culture systems can be maintained. Also,

perfusate can be replaced at rates of up to many volumes per day. Cell retention can thereby be maintained in suspended cell cultures having high cell concentrations. The perfusion rate can also be adjusted by the cell culture system of the present invention to modify cell viability in the system.

The nested-cone separator of the present invention can selectively remove dead cells because of the relatively small size of dead cells and because the Stoke's Law terminal velocity varies inversely with the square of the diameter of cells. Cell

concentration, cell viability, cell retention within the cell culture and cell size within the cell

culture can thereby be controlled by the wide

variations of conditions allowed by the cell culture system of the present invention. The method for separating cells from perfusate of the present

invention thus provides a convenient and

cost-effective way to reliably sustain cell cultures at conditions valuable for production of cells and cell products for prolonged periods of time.

The above features and other details of the invention, either as steps of the method or as

combinations of parts of the invention, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as a limitation of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.

Brief Description of the Drawings

Figure 1 is a side view of one embodiment of the present invention as incorporated into a perfusion cell culture system.

Figure 2 is a sectional view of the embodiment shown in Figure 1.

Figure 3 is a sectional view taken along lines 3-3 of the embodiment shown in Figure 2.

Figure 4 is a sectional view of a second

embodiment of the present invention.

Figure 5 is a sectional view of a third

embodiment of the present invention.

Figure 6 is a sectional view of a fourth

embodiment of the present invention.

Figure 7 is a sectional view of a fifth

embodiment of the present invention.

Figure 8 is a sectional view of a sixth

embodiment of the present invention.

Figure 9 is a plot of cell retention in terms of the fraction of cells retained within perfusate extracted at different superficial velocities

(cm/sec) from two separators of the sixth embodiment of the present invention, having outside diameters of about 62 mm and about 100 mm, as is described in Example 1.

Figure 10 is a plot of cell density (cells/ml) and percent viability in a perfusion cell culture system over time (hours) at a perfusion rate of about 3.5 volumes per day using the fourth embodiment of a cell separator of the present invention, as is described in Example 2.

Figure 11 is a plot of cell density (cells/ml) and percent viability in a perfusion cell culture system over time (hours) at a perfusion rate of about 10 volumes per day using the fourth embodiment of a cell separator of the present invention, as is described in Example 2.

Figure 12 is a plot of cell retention within a perfusate removed from a perfusion cell culture system versus cell size at perfusion rates of about 3.5 and about 10 volumes per day, as is demonstrated in Example 2.

Figure 13 is a plot of cell retention within a perfused cell culture versus cell size at perfusion rates of about 3.5 and about 10 volumes per day, as is demonstrated in Example 2.

Detailed Description of the Invention

A cell culture system 10, shown in Figure 1, supports a perfused or circumfused cell culture 14 within fermenter 16. Cell culture 14 is stirred by impeiler 18 to thereby suspend the cells in cell culture 14. Examples of suitable mammalian cell types for propagation in suspension in the present inventiion include HeLa-S3 (human), BHK (baby hamster kidney) and L cells (mouse), human

lymphoblastoid cells, hybridomas and transfectomas. The present invention is also suitable for culturing bacterial cells, yeast cells, genetically- engineered cells and any other cells that can be cultured in perfusion or circumfusion systems.

Examples of anchorage-dependent cells which can be cultured by the present invention by use of

microcarriers include HeLa (human), 3T3 mouse fibroblasts, mouse bone marrow epithelial cells, Murine Leukemia virus-producing strains of mouse fibroblasts, primary and secondary chick fibroblasts, WI-38 human fibroblast cells, and normal human embryo lung fibroblast cells.

Nested-cone separator 12 has an inlet port 38 which can be fitted to adapter 11 for supporting a Teflon^R polytetrafluoroethylene-coated magnet 13. A motor, not shown, can drive magnet 13 to thereby rotate impeller 18. Perforations 15 at adaptor 11 allow fluid communication between cell culture 14 and inlet port 38. Adapter 11 is submerged in cell culture 14.

As seen in Figure 2, outer cone 30 and inner cone 32 define an annulus 28 to form an expanding cross-sectional area of flow from inlet port 38. Head plate 34 seals annulus 28 from the atmosphere. Outlet port 36 is located in head plate 34 and provides fluid communication between annulus 28 and pump 20, shown in Figure 1. Perfusate and cells from cell culture 14 are drawn into nested-cone separator 12 at inlet port 38 by pump 20. Perfusate is separated from cells in nested-cone separator 12 and is then drawn from nested-cone separator 12 through outlet conduit 22 by pump 20. Cells separate from perfusate in separator 12 and subsequently return to fermenter 16 at inlet port 38. Fresh media is supplied to cell culture 14 through feed conduit 24 from fresh media source 26 to replace perfusate separated from cell culture 14. Inner cone 32 and outer cone 30 are

substantially concentric and form an annulus 28 which can be of essentially constant width, as shown in Figure 3. Outer cone 32 is truncated to form a frustrum of a cone, the narrowest portion of which defines inlet port 38. As perfusate and cells are directed along annulus 28, the path of flow diverges to form an expanding cross-sectional area of flow. Inner cone 30 is truncated to form interior entrance 52. A portion of the perfusate and cells drawn into nested-cone separator 12 from fermenter 16 (Fig. 1) is directed through interior entrance 52 and

interior chamber 40. Perfusate in annulus 28 and in interior chamber 40 combine at head space 42 and are subsequently removed from nested-cone separator 12 through outlet port 36.

Flow of perfusate diverges along annulus 28, whereby velocity of flow of perfusate across inner wall 46 of outer cone 30 diminishes with increasing distance from inlet port 38. The superficial velocity of perfusate within annulus 28 consequently diminishes with increasing distance from inlet port 38. Further, annulus 28 creates a Boycott effect wherein cells settle from the perfusate faster than they would in a vertical tube.

The Stoke's Law terminal velocity at which a cell will settle can be calculated as: where d_p - particle diameter (cm)

Δ_p - density difference (particle-fluid) (g/cm³) g - gravitational acceleration (981 cm/sec²) μ - fluid viscosity (g/cm-sec).

and V_T - terminal velocity (cm/s)

Diminishment of superficial velocity of

perfusate within annulus 28 and the Boycott effect

allow cells to settle across the path of perfusate

flow and to separate from the perfusate. In the

preferred embodiment, the superficial velocity is

reduced within annulus 28 to below the Stoke's Law

terminal velocity of cells to be returned to cell

culture 14. As cells continue to settle, they

collect along inner wall 46 of outer cone 30 and are

directed by inner wall 46 back toward inlet port 38.

Cells 44 collected in close proximity at inner wall

46 can also adhere to each other and their

collective movement can generate a downward current

which entrains other cells settling across annulus

28 to further increase the rate of their separation

from perfusate.

Perfusate from which cells have been separated

in annulus 28 passes through head space 42 (Fig . 2 ) .

Perfus ate within head space 42 is directed toward

outlet port 36. Some fraction of residual cells

carried by the perfusate into head apace 42 further

separate from the perfusate in head space 42 and

settle into interior chamber 40. Perfasate at outlet port 36 is substantially free of cells to be retained within the cell culture at an appropriate perfusion rate. Cells settling from head space 42 mix with cells carried into interior chamber 40 through interior entrance 52. These residual cells separate from perfusate in interior chamber 40 and collect at inner wall 50 of inner cone 32. Inner wall 50 directs separated cells back toward interior entrance 52. Residual cells 48 collected at inner wall 50 can combine with perfusate in annulus 28 at interior entrance 52. These residual cells then separate from perfusate in annulus 28 and collect at inner wall 46 with other cells withdrawn directly from cell culture 14 (Fig. 1). A portion of

residual cells 48 can also return to cell culture 14.

In one alternative embodiment, shown in Figure 4, intermediate cone 54 is nested between outer cone 30 and inner cone 32. Intermediate cone 54 is truncated to form a frustrum of a cone, the

narrowest portion of which defines intermediate entrance 62. Perfusate and cells from perfused cell culture 14 are conducted through intermediate entrance 62 into inner annulus 56 of expanding cross-sectional area for flow. Perfusate flow diverges within inner annulus 56 with increasing distance from intermediate entrance 62. Velocity of perfusate flow across inner wall 60 of

intermediate cone 54 decreases with distance from intermediate entrance 62 such that the superficial velocity of perfusate is diminished and the combination of lowered superficial velocity and the

Boycott effect allows cells which are to be returned to the cell culture to settle across inner annulus 56. In the preferred emodiment of the present invention, the superficial velocity within annulus 56 is diminished to below the Stoke's Law terminal velocity of cells to be returned to the suspended cell culture. Diminished superficial velocity of the perfusate thereby allows cells in the perfusate to separate from the perfusate and to collect along inner wall 60 of intermediate cone 54. Collected cells 58 are directed by inner wall 60 toward intermediate entrance 62.

Cells 58 migrating along inner wall 60 of intermediate cone 54 meet and combine with perfusate in annulus 28 at intermediate entrance 62. A portion of cells 58 can also return to a cell culture through inlet port 52. Operation of

intermediate cone 54 improves the efficiency of nested-cone separator 12 by addition of a second tier of inverted conically shaped flow of perfusate and cells. Perfusate in annulus 28 and inner annulus 56 join at head space 42 where residual cells separate from perfusate in interior chamber 40 for collection along inner wall 50 of inner cone 32. Collected cells 48 are directed by inner wall 50 back to interior entrance 52 and enter flow of perfusate at inner annulus 56. These cells are then separated from perfusate and collect at inner wall 60.

In a third embodiment, shown in Figure 5, perfusate to be separated from a cell culture is directed into nested-cone separator 64 through inlet port 74, defined by outer cone 78, truncated to form a frustrum. Cells and perfusate are conducted along lower annulus 76 of expanding cross-sectional area of flow, which is formed by first outer cone 78 and first inner cone 80. Flow of perfusate within lower annulus 76 diverges and the superficial velocity within lower annulus 76 thereby diminishes as distance from inlet port 74 increases. Superficial velocity of perfusate within lower annulus 76 is thus reduced. In the preferred embodiment, the superficial velocity within lower annulus 76 is reduced to below that of the Stoke's Law terminal velocity of the cell to be returned to the cell culture. A Boycott effect and diminished

superficial velocity allows cells to settle across lower annulus 76 and to collect along inner wall 68 of outer cone 78. Collected cells 66 are directed by inner wall 68 back toward inlet port 74 for re-entry to the perfused cell culture.

Cells within the perfusate which do not collect along inner wall 68 are conducted by the perfusate into upper annulus 82, which forms a converging cross-sectional area of flow. Flow of perfusate in upper annulus 82 is formed by second inner cone 70 and second outer cone 86, which are inverted with respect to first inner cone 80 and first outer cone 78. Second inner cone 70 and second outer cone 86 are advantageous because they can provide for a substantially constant rate of acceleration to minimize turbulence of perfusate as it approaches exit port 90 and can continue separation of cells from perfusate within nested-cone separator 64.

Thus, cells which do not separate from perfusate at lower annulus 76 can settle across the width of perfusate flow within upper annulus 82. These remaining cells, which are carried from lower annulus 76 into upper annulus 82, can collect at wall 84 of second inner cone 70. Collected cells 88 at wall 84 can adhere to each other and entrain cells settling across the width of upper annulus 82. Lower cone 80 and upper cone 70 meet to form lip 72. At lip 72, cells 88 are directed by wall 84 into lower annulus 76. Cells from upper annulus 82 then separate from perfusate within lower annulus 76 and collect along inner wall 68 of first outer cone 78. Collected cells 66 are directed by inner wall 68 back toward inlet port 74 to re-enter the suspended cell culture. Substantially cell-free perfusate is conducted through outlet port 90 for further

processing.

Figure 6 illustrates a fourth embodiment of the invention, in which an intermediate cone 92 can be disposed between outer cone 78 and inner cone 80. Two annuli forming expanding cross-sectional areas of flow will thus be formed. Intermediate cone 92, truncated to form a frustrum, defines interior entrance 96 at its narrowest point. Collected cells 88 at wall 84 of upper cone 70 can enter interior annulus 94 at lip 72. Cells within interior annulus 94 settle across perfusate flow and collect at inner wall 102. A portion of collected cells 100 can return to the perfused cell culture. Collected cells 100 can also combine with perfusate entering lower annulus 76 at interior entrance 96. Cells collected from upper annulus 82 are thereby

introduced to perfusate in lower annulus 94 at lip 72. The efficiency of nested-cone separator 64 is somewhat enhanced by delivering collected cells at the entrance of lower annulus 94.

In a fifth embodiment, shown in Figure 7, nested-cone separator 104 can be partially submerged at outer cone 106 in a perfused cell culture. Inner cone 108 is nested within outer cone 106. Outer cone 106 and inner cone 108 define annulus 110, thereby forming an expanding cross-sectional area of flow with increasing distance from the suspended cell culture. Perfusate from the suspended cell culture is drawn into annulus 110 at inlet port 112, defined by the narrowest portion of outer cone 106, truncated to form a frustrum. Inner cone 108 is truncated to form a frustrum, the narrowest portion of which defines interior entrance 126. A portion of perfusate and cells can enter head space 118 at interior entrance 126. Cells separate from the perfusate and collect along inner wall 116 of outer cone 106. Collected cells 114 are directed by inner wall 116 back toward inlet port 112. Perfusate is directed from annulus 110 to head space 118, which is defined by inner cone 108 and upper cone 120.

Residual cells in the head space 118 separate from perfusate and collect along inner wall 122 of inner cone 108. Collected cells 124 are directed by inner wall 122 toward interior entrance 126 where a portion of the cells enter annulus 110. A portion of cells 124 can also return to the suspended cell culture. Substantially cell-free perfusate is removed from nested-cone separator 104 at outlet port 128.

In a sixth embodiment of the present invention, an intermediate cone 130 can be disposed between outer cone 106 and inner cone 108, as shown in

Figure 8. Collected cells 124 at inner wall 122 of interior cone 108 collect along inner wall 122 and combine with perfusate at intermediate entrance 138. Intermediate cone 130 is truncated to form a

frustrum and thereby defines intermediate entrance 138. Collected cells 134 are directed by inner wall 136 toward intermediate entrance 138 and combine with perfusate entering annulus 110. A portion of collected cells 124 and 134 can re-enter the

suspended cell culture through inlet port 112.

Efficiency of nested-cone separator 104 is improved by intermediate cone 130 by reducing the distance across which cells must settle, by increasing the surface area per volume of flow of the perfusate, and by delivery of cells collected in interior annulus 132 to the entrance of an adjacent annulus.

Example 1

Two nested-cone separators, of the embodiment shown in Figure 8, were placed in a 250 ml fermenter containing a suspension of TK6 human B-lymphoblastoid cells at a density of about 10 cells/ml. One separator had an outside diameter of about 62 mm and a distance between the outer cone, the inner cone, and the intermediate cone of about 1/8 inches. The other separator had an outside diameter of about 100 mm and distances between the outer cone, the inner cone, and the intermediate cone of about 1/4 inches. The annuli of flow between cones of both separators had an angle of flow relative to the horizontal of about 60°. Calibrated pumps, fitted to the

separators, withdrew perfusate from the perfused cell cultures at flow rates of between about 0.8 - 5.2 liters per day for the smaller separator, and between about 4 - 20 liters per day for the larger separator. A Coulter counter was used to measure the concentration of cells in perfusate exiting the separators. The cells were approximately 12 μm in diameter and had terminal settling velocities of about 3.9 X 10 ^-4 cm/sec. Figure 9 is a plot of separation efficiency, ε, which is the ratio of cell concentration exiting the top of the separator to the cell concentration of perfusate at the entrance of the separator. As can be seen in Figure 9, both the larger and the smaller nested-cone separators exhibited cell loss in perfusate of less than 20% in a perfusion cell culture system having a superficial velocity of between up to about 8 x 10 ^-4 cm/sec.

Example 2

A nested-cone separator, as is shown in Figure 6, was placed in a perfusion cell culture of

Sp2/0-derived transfectoma cells having a volume of 250 ml and an initial cell concentration of about 9 x 10⁵ cell/ml. The distances between the inner cone, the outer cone, and the intermediate cone were about 1/8 inches. The inlet port and the outlet ports were about 1/2 inches in diameter. The lower annulus and the interior annulus of flow had an angle relative to the horizontal of about 60°. The inlet and outlet ports had diameters of about 1/2 inches.

Figure 10 shows the growth rate and viability over time (days) of the cell culture at a perfusion rate of about 10 volumes per day. Maximum cell concentration was about 2 X 10⁷ cell/ml. Cell concentration was maintained over 1 X 10⁷ cells/ml for a period of about a week. Figure 11 shows the growth rate and viability over time (days) of the cell culture at a perfusion rate of about 3.5 volumes per day. Maximum cell concentration was about 1 X 10⁷ cells/ml for a period of about a week. Cell viability of the cell culture at a perfusion rate of 10 volumes per day was above about 80% for more than 16 days. Cell viability at a perfusion rate of about 3.5 volumes per day was above about 70% for more than about 11 days. Thus, it can be seen that cell concentration and cell viability can be controlled by the present invention by adjusting the perfusion rate of the cell culture.

Cell loss was measured as a function of cell diameter for perfusion rates of about 3.5 and about 10 volumes per day, and is shown in Figure 12.

When the perfusion rate was about 3.5 volumes per day, the distribution of cell sizes retained within the perfusate was different than was demonstrated at a perfusion rate of about 10 volumes per day.

Therefore, it can be seen that the distribution of cell sizes removed from a perfused cell culture can be modified by changing the perfusion rate.

Further, as can be seen in Figure 13, the

distribution of cell sizes in the suspended cell culture differed at perfusion rates of about 3.5 and about 10 volumes per day. Thus, the distribution of cell sizes in the perfused cell culture can also be modified by changing the perfusion rate in a

suspended cell culture system. Equivalents

It is to be understood that the cones of the present invention need not be concentric and that the annuli of flow of the present invention can deviate in width between the inlet port and outlet port without departing from the scope of the

invention. It is also to be understood that "cones" as that term is used to describe the present

invention includes pyramids and prismatoids having three or more faces, and that the pyramids and prismatoids can be truncated to form frustrums, the narrowest portions of which defining inlet ports, interior entrances and intermediate entrances.

Further, additional cones can be interposed between existing cones of the nested-cone separator to form additional expanding cross-sectional areas of flow and greater capacity for separation of perfusate from cells. The number of cones and the rate of flow can be modified for control of perfused and circumfused cell cultures.

It is also to be understood that the present invention can be used to separate suspended solid particles or immiscible liquids from fluids in other operating systems, e.g., for separation of solids from liquids in suspensions and colloids.

Although preferred embodiments have been specifically described and illustrated herein, it will be appreciated that many modifications and variations of the present invention are possible, in light of the above teachings, within the purview of the following claims, without departing from the spirit and scope of the invention.

Claims

1. A cell culture system wherein perfusate is

separated from cells during removal of

perfusate from a cell culture vessel and wherein the cells are collected and directed back to the cell culture vessel, the cell culture system comprising:

a cell culture vessel containing a medium and cells;

a nested-cone separator having an outer cone and an inner cone, the distance between the outer cone and the inner cone defining an annulus which forms an expanding

cross-sectional area of flow from an inlet port to an outlet port, which is elevated from the inlet port, the narrowest portion of the outer cone defining an inlet port through which perfusate and cells can be withdrawn from the cell culture vessel into the annulus, whereby cells are separated from perfusate in the annulus and returned at the inlet port to the culture vessel;

means for withdrawing perfusate and cells from the cell culture vessel through the nested-cone separator, whereby perfusate is removed from the cell culture system through the outlet port at the nested-cone separator; and

means for introducing fresh medium into the cell culture vessel.

2. A nested-cone separator for separating

perfusate from cells during removal of

perfusate from a cell culture vessel and wherein the separated cells are collected and directed back to a cell culture vessel, the nested-cone separator comprising:

an outer cone and an inner cone nested within the outer cone to define an annulus with an expanding cross-sectional area for flow of perfusate from a cell culture;

an inlet port, defined by a narrow portion of the outer cone, through which perfusate can be withdrawn from the cell' culture into the annulus and for return of cells from the annulus to the cell culture;

an interior entrance defined by a narrow portion of the inner cone; and

an outlet port elevated above the inlet port for removal of perfusate from the cell culture.

3. The nested-cone separator of Claim 2 wherein the inner cone is substantially concentric to the outer cone.

4. The nested-cone separator of Claim 2 further comprising at least one intermediate cone disposed between the outer cone and the inner cone, and wherein the intermediate cone has an intermediate entrance defined by a narrow portion of the intermediate cone.

5. A nested-cone separator wherein perfusate is separated from cells during removal of

perfusate from a cell culture vessel and wherein the cells are collected and directed back to the cell culture vessel, the nestedcone separator comprising:

a first outer cone and a first inner cone nested within the first outer cone to define a lower annulus with an expanding cross-sectional area for flow of perfusate from a cell culture; an inlet port, defined by a narrow portion of the first outer cone, through which

perfusate can be withdrawn from the cell culture into the annulus and for return of cells from the annulus to the suspended cell culture; and

6. The nested-cone separator of Claim 5 wherein the first inner cone is substantially

concentric with the first outer cone.

7. The nested-cone separator of Claim 5 further comprising a second inner cone inverted with respect to the first inner cone, which is of equal base diameter to the first inner cone and which is adjacent to the first inner cone at the base of the first inner cone.

8. The nested-cone separator of Claim 7 further comprising a second outer cone adjoining the first outer cone, the second outer cone and second inner cone defining an annulus, thereby forming a converging cross-sectional area for flow of perfusate through the upper annulus.

9. The nested-cone separator of Claim 5 further comprising at least one intermediate cone disposed between the first outer cone and the first inner cone, and wherein the intermediate cone has an intermediate entrance defined by a narrow portion of the intermediate cone.

10. A method for separation of perfusate from a

cell culture comprising the steps of directing perfusate and cells from the cell culture between an outer cone and an inner cone

defining an annulus, thereby forming an

expanding cross-sectional area of flow for reducing the velocity of the perfusate flow within the annulus, thereby allowing cells to settle across path of perfusate flow through the annulus and to collect at an inner wall of the outer cone, whereby perfusate is separated from cells and is removed from the cell culture while cells which have been separated from the perfusate are directed by the inner wall back to the cell culture.

11. A method for separation of perfusate from a cell culture comprising the steps of directing perfusate and cells from the cell culture between a first outer cone and a first inner cone which defines an lower annulus and thereby forms an expanding cross-sectional area of flow for reducing the superficial velocity of the perfusate flow within the lower annulus, thereby allowing cells to settle across the lower annulus and to collect at an inner wall of the first outer cone, whereby perfusate is separated from cells and is removed from the cell culture while cells which have been separated from the perfusate are directed by the inner wall back to the cell culture.

12. The method of Claim 11 further comprising the steps of forming an upper annulus of perfusate flow defined by a second inner cone and a second outer cone, thereby forming a converging cross-sectional area for flow of perfusate through the upper annulus, the base of the second inner cone adjoining the base of the first inner cone and being of equal base diameter to and inverted with respect to the first inner cone, and the base of the second outer cone adjoining the base of the first outer cone and being of equal base diameter to and inverted with respect to the first outer cone, separating residual cells from the perfusate at the upper annulus, and directing residual cells along a wall of the second inner cone toward the lower annulus for settling across the path of perfusate flow through the lower annulus, whereby cells return to the cell culture.

13. A method of Claim 11 further comprising the

steps of disposing an intermediate cone between the first inner cone and the first outer cone, thereby defining an Interior annulus forming an expanding cross-sectional area of flow for separating living cells from perfusate and thereby allowing such cells to settle across the path of perfusate flow through the interior annulus and to collect along an inner wall of the intermediate cone, whereby the intermediate cone directs the collected cells back to the cell culture.

14. A nested-cone separator for separating a liquid from solids during removal of the liquid from a suspension vessel and wherein the solids are collected and redirected back to the

suspension, the nested-cone separator comprising:

an outer cone and an inner cone nested within the outer cone defining an annulus with an expanding cross-sectional area of flow for separating liquid from the suspension; an inlet port, defined by a portion of the outer cone, through which the suspension can be drawn into the annulus for separation of liquid from the solids and for return of the solids from the annulus to the suspension vessel;

an interior entrance defined by a portion of the inner cone; and

an outlet port elevated above the inlet port for delivering the liquid from the

nested-cone separator.

15. A nested-cone separator for separating first liquid from a second liquid which is

substantially immiscible in the first liquid, during removal of the first liquid from a colloid and wherein the second liquid is collected and directed back to the colloid, the nested-cone separator comprising:

an outer cone and an inner cone nested within the outer cone to define an annulus with an expanding cross-sectional area for flow of the first liquid from the colloid;

an inlet port, defined by a portion of the outer cone, through which the colloid can be drawn into the annulus for separation of first liquid from the second liquid and for return of the second liquid from the annulus to the colloid vessel;

an interior entrance defined by a portion of the inner cone; and

an outlet port elevated above the inlet port for delivering the first liquid from the nested-cone separator.

16. A nested-cone separator for maximizing cell viability within a cell culture and for separating perfusate from cells during removal of perfusate from a cell culture vessel, wherein the separated cells are collected and directed back to a cell culture, the

nested-cone separator comprising:

an outer cone and an inner cone nested within the outer cone to define an annulus, thereby forming an expanding cross-sectional area for flow of perfusate from the cell culture;

an inlet port, defined by a narrow portion of the outer cone, through which perfusate can be withdrawn from the cell culture into the annulus and for return of cells from the annulus to the suspended cell culture;

an interior entrance defined by a narrow portion of the inner cone; and

17. A method for producing cell growth by-products a) inoculating a medium with cells to form a cell culture;

b) directing perfusate and cells from the

cell culture between an outer cone and an inner cone defining an annulus, thereby forming an expanding cross-sectional area of flow for reducing the velocity of the perfusate flow within the annulus, thereby allowing cells to settle across the path of perfusate flow through the annulus and to collect at an inner wall of the outer cone, whereby perfusate is separated from cells and is removed from the cell culture while cells which have been separated from the perfusate are directed by the inner wall back to the cell culture;

c) maintaining the cell culture under

conditions conducive to the produciton of cell growth by-products; and

d) harvesting the cell growth by-products.

18. The method of Claim 17 wherein the cell growth by-product comprises virus.

19. The method of Claim 17 wherein the cell growth by-product comprises monoclonal antibodies.

20. The method of Claim 17 wherein the cell growth by-product comprises hormones.

21. The method of Claim 17 wherein the cell growth by-product comprises cell-derived

macromolecules.