The culturing of living cells in vitro is performed for a variety of purposes, including the preparation of viral vaccines, the recovery of valuable by-products of cell metabolism and the production of tissue-like derivatives for creating artificial organs.
Several problems are associated with growing living cells in vitro to produce dense masses of cells. First, individual components of the nutrient medium must diffuse through the cell layers to reach all cells. This becomes increasingly difficult as the thickness of the cell layer increases.
Second, the maintenance of a suitable environment for cell growth is difficult because the fluid immediately adjacent a growing cell is continuously changing as cellular metabolism proceeds and is returned to its original status only in stepwise fashion when the nutrient medium is changed or agitated en masse.
Third, a lattice or suitable material upon which to grow some types of cells is required.
Various types of apparatus and methods have been developed in response to these needs. One method involves attaching and growing cells on the interior surface of plastic or glass roller tubes and bottles as disclosed in U.S. Pat. No. 3,450,598. Another method involves attaching the cells to a flat surface of stationary containers such as petri dishes or rectangularly shaped culture plates. The flat surfaces can be stacked one on top of each other in a spaced-apart array as disclosed in U.S. Pat. No. 3,843,454.
The use of hollow fibers or synthetic capillaries has more recently been disclosed as a support matrix for the propagation of cells. For example, U.S. Pat. Nos. 3,821,087; 3,883,393; 4,184,922; 4,200,689; 4,206,015 and 4,220,725, all to Knazek et al, variously disclose apparatus and methods for the in vitro growth of cells on semi-permeable, tubular membranes or capillaries wherein cells are initially allowed to settle onto the outer surfaces of the capillary walls in a nutrient medium. Nutrients diffuse from the perfusing medium through the capillary walls and are utilized by the cells. Cell products diffuse from the cells, through the capillary walls and into the perfusate, from which cell products may be recovered.
U.S. Pat. Nos. 4,184,922 and 4,200,689 disclose cell culturing devices comprising a single bundle of fibers wherein some of the fibers are connected to one perfusion circuit and the remaining fibers are connected to a second perfusion circuit. The difference in pressure between the two circuits produces convective currents of perfusate within the extracapillary space and thereby improves nutrient distribution to the growing cells.
In U.S. Pat. No. 4,220,725, a bundle of capillaries, upon which cells are allowed to grow, is wrapped in a porous envelope or sheet material which creates an extra-envelope space into which the cells can migrate for periodic removal without disturbing the main cell culture. The creation of the extra-envelope space increases the surface area for nutrient end waste product diffusion to and from the cells located on the outer surface of the capillaries.
In U.S. Pat. No. 3,997,396, cells are attached to and grown on one side or surface of a single hollow fiber membrane wherein the cells are propagated and maintained by passing oxygen through the membrane from the side opposite that to which the cells are attached and into contact with the cells while simultaneously incubating the cells in a nutrient medium. By continuously passing oxygen through the membrane from the side opposite that on which the cells are attached, a continuous and uniform supply of oxygen reaches and nourishes the cells thereby facilitating aerobic propagation of the cells in the desired tissue densities.
In U.S. Pat. Nos. 4,087,327 and 4,201,845 to Feder et al, an in vitro cell culture reaction system is disclosed which utilizes elongate hollow or solid fibers arranged in a shallow layer configuration as a matrix for cell attachment on the outer surface of the fibers. Nutrient media flow is directed substantially uniformly through the fiber layer and substantially transverse to the plane of the elongate axes of the fibers. The cells are aerated by passing oxygen through the interior of the fibers which then permeates the fiber walls. The use of a shallow bed of fibers in a relatively short path of media flow results in a substantial reduction of the nutrient and metabolic product gradients that is normally produced by the fibrous bundle as well as a more extensive utilization of the fiber surface for cell attachment.
U.S. Pat. No. 4,391,912 discloses a device for cultivating floating animal cells comprising a gas permeable shell and a plurality of hollow fibers enclosed within the shell, wherein the hollow fibers are open at either end outside of the shell and have a pore diameter of from about 102 angstroms to 5.times.10.sup.4 angstroms. Nutrient medium passes through the interior of the hollow fibers and oxygen passes through the shell and the animal cells are cultivated in the space between the shell and the hollow fibers. These pore diameters of the hollow fibers are disclosed as optimizing efficient exchange of nutrients and metabolic products produced by the cells resulting in high density cell growth.
Notwithstanding the usefulness of the hollow fiber cell culture devices, it has been found that the nutrient media flow through the hollow capillaries prevents complete penetration of the capillary bundle by the cells and sets up an undesirable gradient of medium flow. As a result, there is an incomplete utilization of the available capillary surface for cell attachments and cells become unevenly distributed along the surface. Also, as the nutrient medium flows through the reactor, nutrients are more available to the cells near the inlet, and as the medium flows to the outlet, metabolic products such as lactic acid accumulate in the medium, undesirably affecting pH and producing other toxic effects on the cells.
Another significant difficulty encountered with these hollow fiber-type cell culture devices concerns the high media circulation rates necessary to supply adequate oxygen to the cells. Specifically, aqueous nutrient media, equilibrated with air, is able to carry 4.5 ml of oxygen per liter (37.degree. C., 760 mm of Hg). This relative inability of aqueous solutions to carry oxygen causes the rate at which oxygen is supplied to the cells to be the limiting step in in vitro cell growth operations. In order to produce high yields of cells and/or cell secreted products, media circulation rates must be increased to provide more oxygen to cells. High circulation rates in turn cause high internal pressure and turbulence which has presented problems in terms of constructing the device on an industrial scale and in propagating mammalian cells whose sensitivity and fragility prohibit the use of too vigorous aeration and/or agitation. Vigorous aeration also causes the denaturation of many biologically and medicinally useful proteins produced by cell cultures.
Moreover, the above-described hollow fiber-type devices which provide for separate oxygen and nutrient media delivery to cells suffer from the additional disadvantages of being mechanically complex, difficult to assemble and being unduly large. Moreover, the dimensions of these devices are not constrained to maintain the growing cells in close proximity to the nutrient media supply source thus causing undesirable nutrient gradients.
Therefore, it has been desirable to provide new cell culturing devices for growing cells in vitro, particularly mammalian cells, which overcome the various difficulties associated with the prior art devices and produce cells and/or cell secreted products more economically and in higher yields.